Integrated purification and measurement of dna methylation and co-measurement of mutations and/or mrna expression levels in an automated reaction cartridge

ABSTRACT

Methods of determining methylation of DNA are provided. In one illustrative, but non-limiting embodiment the method comprises i) contacting a biological sample comprising a nucleic acid to a first matrix material comprising a first column or filter where said matrix material binds and/or filters nucleic acids in said sample and thereby purifies the DNA; ii) eluting the bound DNA from the first matrix material and denaturing the DNA to produce eluted denatured DNA; iii) heating the eluted DNA in the presence of bisulfite ions to produce a deaminated nucleic acid; iv) contacting said deaminated nucleic acid to a second matrix material comprising a second column to bind said deaminated nucleic acid to said second matrix material; v) desulphonating the bound deaminated nucleic acid and/or simultaneously eluting and desulphonating the nucleic acid by contacting the deaminated nucleic acid with an alkaline solution to produce a bisulfite converted nucleic acid; vi) eluting said bisulfite converted nucleic acid from said second matrix material; and vii) performing methylation specific PCR and/or nucleic acid sequencing, and/or high resolution melting analysis (HRM) on said bisulfite-converted nucleic acid to determine the methylation of said nucleic acid, wherein at least steps iv) through vi) are performed in a single reaction cartridge.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of prior U.S. provisionalapplication No. 62/433,165, filed Dec. 12, 2016, which is herebyincorporated by reference in its entirety.

BACKGROUND

The genomes of higher eukaryotes contain the modified nucleoside5-methyl cytosine (5-meC). This modification is usually found as part ofthe dinucleotide CpG in which cytosine is converted to 5-methylcytosinein a reaction that involves flipping a target cytosine out of an intactdouble helix and transfer of a methyl group from S-adenosylmethionine bya methyltransferase enzyme (see, e.g., Klimasauskas et al. (1994) Cell76: 357-369). This enzymatic conversion is the primary epigeneticmodification of DNA known to exist in vertebrates and is essential fornormal embryonic development (see, e.g., Bird (1992) Cell 70: 5-8; Lairdand Jaenisch (1994) Human Mol. Genet. 3: 1487-1495; and Li et al. (1992)Cell 69: 915-926).

In eukaryotes, DNA methylation regulates normal cellular processes suchas genomic imprinting, chromosomal instability, and X-chromosomeinactivation. Typically, DNA methylation occurs at the fifth carbonposition of cytosine at dinucleotide 5′-CpG-3′ sites in or near genepromoters termed CpG islands or shores. Methylation controls geneexpression by down-regulating transcription either by directlyinhibiting transcriptional machinery or indirectly through therecruitment of chromatin remodeling proteins. Chromosomal methylationpatterns change dynamically during embryonic development, and thecorrect methylation patterns have to be maintained throughout anindividual's lifetime. Changes in methylation patterns are linked toaging, and errors in DNA methylation are among the earliest changes thatoccur during oncogenesis. Thus, the detection of methylation at genepromoters is important, inter alia, for diagnosing and/or monitoringpatients with cancer.

Epigenetic alterations, including DNA methylation, interrupt theDNA-RNA-protein axis which describes how genetic information istranscribed into messenger RNAs (mRNAs). The correlation between genomicDNA variation, mRNA copy numbers and protein levels may be described byDNA methylation levels. Thus co-measurement of DNA methylation levelsand corresponding down-stream mRNA levels can be important tounderstanding the mechanism of epigenetic cellular regulation.

Several methods have been developed to detect and quantify methylationefficiently and accurately. The most common technique is the bisulfiteconversion method which converts unmethylated cytosines to uracil. Onceconverted, the methylation profile of DNA can be determined by standardPCR techniques, sequencing methods, and the like.

There are several DNA Methylation kits suitable for bisulfite conversionand DNA cleanup (e.g., EZ DNA METHYLATION™ kits from Zymo Research).Most kits involve several steps, reagents, and incubation times andoften require purified DNA before conversion although some kits canutilize tissue or plasma/serum as starting material.

Typically the bisulfite conversion process requires at least foursteps: 1) DNA Denaturation; 2) Bisulfite Incubation; 3) DNAPurification; and 4) Desulphonation. The final desulphonation step canbe completed on-column or in solution followed by an ethanolprecipitation. There are currently no methylation kits that allow a userto complete the entire process—DNA purification, bisulfite incubation,desulphonation, second DNA purification, and methylation-specific PCRall in one step.

SUMMARY

Various embodiments contemplated herein may comprise, but need not belimited to, one or more of the following:

Various embodiments contemplated herein may include, but need not belimited to, one or more of the following:

Embodiment 1

A method of determining the methylation state of a nucleic acid, saidmethod comprising:

i) contacting a biological sample comprising a nucleic acid to a firstmatrix material comprising a first column or filter where said matrixmaterial binds and/or filters nucleic acids in said sample and therebypurifies the DNA;

ii) eluting the bound DNA from the first matrix material and denaturingthe DNA to produce eluted denatured DNA;

iii) heating the eluted DNA in the presence of a bisulfite reagent toproduce a deaminated nucleic acid;

iv) contacting said deaminated nucleic acid to a second matrix materialcomprising a second column to bind said deaminated nucleic acid to saidsecond matrix material;

v) desulphonating the bound deaminated nucleic acid and/orsimultaneously eluting and desulphonating the nucleic acid by contactingthe deaminated nucleic acid with an alkaline solution to produce aconverted (e.g., bisulfite converted) nucleic acid;

vi) eluting said bisulfite converted nucleic acid from said secondmatrix material; and

vii) performing methylation specific PCR and/or nucleic acid sequencing,and/or high resolution melting analysis (HRM) on said converted nucleicacid to determine the methylation of said nucleic acid, wherein at leaststeps iv) through vi) are performed in a single reaction cartridge.

Embodiment 2

The method of embodiment 1, wherein at least steps iv) through vi) areperformed in a single reaction cartridge.

Embodiment 3

The method of embodiment 1, wherein at least steps iii) through vi) areperformed in a single reaction cartridge.

Embodiment 4

The method of embodiment 1, wherein at least steps ii) through vi) areperformed in a single reaction cartridge.

Embodiment 5

The method of embodiment 1, wherein at least steps i) through vi) areperformed in a single reaction cartridge.

Embodiment 6

The method according to any one of embodiments 1-5, wherein step vii isperformed in the same reaction cartridge.

Embodiment 7

The method according to any one of embodiments 1-6, wherein said firstmatrix material and said second matrix material are the same materialforming the same column.

Embodiment 8

The method according to any one of embodiments 1-7, wherein said firstmatrix material and said second matrix material form different columns.

Embodiment 9

The method according to any one of embodiments of embodiment 1-8,wherein said methylation specific PCR, when performed, is performed insaid cartridge.

Embodiment 10

The method according to any one of embodiments 1-9, wherein said nucleicacid sequencing, when performed, is performed in said cartridge or in adevice coupled to said cartridge.

Embodiment 11

The method according to any one of embodiments 1-10, wherein saidcartridge comprises a column comprising said first matrix material, asample receiving chamber, a temperature controlled channel or chamber, aplurality of chambers containing reagents and/or buffers, and when inuse at least one of said chambers contains a desulphonation/elutionbuffer, and wherein said cartridge optionally comprises a second columncomprising said second matrix material.

Embodiment 12

The method of embodiment 11, wherein, when in use, at least one of saidchambers contains a reagent that provides bisulfite ions.

Embodiment 13

The method according to any one of embodiments 11-12, wherein saidsecond column is absent.

Embodiment 14

The method according to any one of embodiments 11-13, wherein saidsecond column is present.

Embodiment 15

The method according to any one of embodiments 11-14, wherein saidcartridge comprises a thermocycling channel or chamber in addition tosaid temperature controlled channel or chamber.

Embodiment 16

The method according to any one of embodiments 11-14, wherein saidtemperature controlled channel or chamber is a thermocycling channel orchamber.

Embodiment 17

The method according to any one of embodiments 11-16, wherein saidcartridge comprises one or more chambers containing one or more reagentsselected from the group consisting of methylation specific PCR primers,methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer.

Embodiment 18

The method of embodiment 17, wherein said cartridge comprises one ormore chambers containing one or more primers and probes for detection ofmethylation of a forward strand of a bisulfite-converted DNA.

Embodiment 19

The method according to any one of embodiments 17-18, wherein saidcartridge comprises one or more chambers containing one or more primersand probes for detection of methylation of a reverse strand of abisulfite-converted DNA.

Embodiment 20

The method according to any one of embodiments 11-19, wherein saidsample receiving chamber, said column(s), said plurality of chambers,and when present, said temperature controlled channel or chamber and/orthermocycling channel or chamber, are selectively in fluidcommunication.

Embodiment 21

The method of embodiment 20, wherein said sample receiving chamber, saidcolumn(s), said plurality of chambers, and when present, saidthermocycling channel or chamber, are selectively in fluid communicationby microfluidic channels and valves.

Embodiment 22

The method of embodiment 20, wherein said sample receiving chamber, saidcolumn(s), said plurality of chambers, and when present, saidthermocycling channel or chamber or a port into said thermocyclingchannel or chamber, are disposed around a central valve and selectivelyin fluid communication with a channel in said central valve, whereinsaid central valve is configured to accommodate a plunger that iscapable of drawing fluid into or out of a chamber in fluid communicationwith said central valve.

Embodiment 23

The method according to any one of embodiments 11-22, wherein saidcartridge, when in use, comprises:

a first chamber containing a sample;

a second chamber containing a guanidinium thiocyanate-ethanol (GTC-EtOH)solution;

a third chamber containing a bisulfite reagent;

a fourth chamber containing a buffer;

a fifth chamber containing a rinse solution; and

a sixth chamber containing an elution/desulphonation reagent.

Embodiment 24

The method of embodiment 23, wherein first chamber contains said samplein a GTC-EtOH-Tween extraction/precipitation reagent.

Embodiment 25

The method according to any one of embodiments 23-24, wherein theGTC-ETOH-Tween buffer is added at or near the time the sample is placedinto the cartridge.

Embodiment 26

The method according to any one of embodiments 23-25, wherein thebisulfite reagent is added to the cartridge at or near the time thesample is placed in the cartridge.

Embodiment 27

The method of embodiment 23, wherein the GTC-ETOH-Tween buffer isprovided as a component of the cartridge.

Embodiment 28

The method according to any one of embodiments 23-25, wherein thebisulfate reagent is provided as a component of the cartridge.

Embodiment 29

The method according to any one of embodiments 11-28, wherein saidcartridge comprises a seventh chamber containing PCR primers and/orprobes and/or PCR enzymes.

Embodiment 30

The method according to any one of embodiments 11-29, wherein saidcartridge comprises an eighth chamber also containing PCR primers and/orprobes and/or PCR enzymes.

Embodiment 31

The method of embodiments 29-30, wherein said PCR primers, and/orprobes, and/or enzymes are provided as beads.

Embodiment 32

The method according to any one of embodiments 1-31, wherein saidbiological sample comprises one or more samples selected from the groupconsisting of a cell, a tissue, and a biological fluid containing anucleic acid.

Embodiment 33

The method of embodiment 32, wherein said biological sample comprises abiological fluid selected from the group consisting of whole blood,plasma, serum, saliva, mucus, urine, sputum, pancreatic juice, andcerebrospinal fluid.

Embodiment 34

The method of embodiment 32, wherein said biological sample comprises asample selected from the group consisting of a tissue sample, a formalinfixed paraffin embedded (FFPE) tissue, fresh frozen tissue, fine needleaspirates (FNA), and a core biopsy.

Embodiment 35

The method according to any one of embodiments 1-34, wherein said methodcomprises contacting said biological sample with a lysis solution.

Embodiment 36

The method of embodiment 35, wherein said method comprises providingsaid sample in said sample receiving chamber and contacting said samplewith an extraction/precipitation solution.

Embodiment 37

The method according to any one of embodiments 1-36, wherein said matrixmaterial comprises a column material selected from the group consistingof glass or silica, an ion exchange resin, cellulose, andhydroxyapatite.

Embodiment 38

The method of embodiment 37, wherein said matrix material comprisesglass.

Embodiment 39

The method according to any one of embodiments 1-38, wherein saidbisulfite ion is provided as compound selected from the group consistingof ammonium bisulfite, sodium metabisulfite, potassium bisulfite, cesiumbisulfite, and DABSO.

Embodiment 40

The method of embodiment 39, wherein said bisulfite ion is provided byammonium bisulfite.

Embodiment 41

The method according to any one of embodiments 1-40, wherein saidbisulfite is provided in a reagent mix comprising scavengers to preventsulfite oxidation and/or catalysts.

Embodiment 42

The method of embodiment 41, wherein said bisulfite is provided in areagent mix comprising scavengers selected from the group consisting ofTrolox and hydroquinone.

Embodiment 43

The method according to any one of embodiments 41-42, wherein saidbisulfite is provided in a reagent mix comprising polyamines ascatalysts.

Embodiment 44

The method according to any one of embodiments 1-43, wherein saideluting the bound DNA comprises eluting and denaturing said DNA using alow concentration of potassium hydroxide or other base.

Embodiment 45

The method of embodiment 44, wherein said eluting the bound DNAcomprises eluting and denaturing said DNA with an alkaline solution witha pH greater than about pH 10.5.

Embodiment 46

The method of embodiment 44, wherein said eluting the bound DNAcomprises eluting and denaturing said DNA with an alkaline solution witha pH greater than about pH 12.

Embodiment 47

The method of embodiments 45-46, wherein said alkaline solution is a10-15 mM KOH solution.

Embodiment 48

The method according to any one of embodiments 1-47, wherein saidincubating the eluted DNA with bisulfite ions to produce a deaminatednucleic acid comprises incubating the DNA in an ammonium bisulfitesolution having a concentration that ranges from about 6M to about 7M.

Embodiment 49

The method of embodiment 48, wherein said incubating the eluted DNA withbisulfite ions to produce a deaminated nucleic acid comprises incubatingthe DNA in an ammonium bisulfite solution having a concentration ofabout 6.5M.

Embodiment 50

The method of embodiment 49, wherein said incubating comprisestransferring the DNA in a concentrated bisulfite solution into atemperature controlled channel or chamber in said cartridge and heatingsaid mixture.

Embodiment 51

The method of embodiment 50, wherein said incubating comprises thermallycycling the concentrated bisulfite solution from a temperature of about60° C. to about 95° C.

Embodiment 52

The method according to any one of embodiments 1-51, wherein saidcontacting said deaminated nucleic acid to a second matrix materialcomprises mixing the DNA-bisulfite solution with fresh GTC-EtOH anddispensing the solution over said second matrix material.

Embodiment 53

The method of embodiment 52, wherein said method comprises washing theDNA in said second matrix material with fresh GTC-EtOH, and then a rinsesolution.

Embodiment 54

The method of embodiment 53, wherein said rinse solution comprisesPEG200.

Embodiment 55

The method according to any one of embodiments 1-54, wherein saiddesulphonating the bound deaminated nucleic acid comprises eluting theDNA from said second column with a high pH desulphonation buffer andincubating said solution.

Embodiment 56

The method of embodiment 55, wherein said incubating is for a period oftime ranging from about 1 minute to about 1 hour, or from about 5minutes to about 30 minutes, or from about 10 minutes to about 20minutes, or for about 15 minutes.

Embodiment 57

The method of embodiments 55-56, wherein said high pHdesulphonation/elution buffer comprises KOH.

Embodiment 58

The method according to any one of embodiments 55-57, wherein saidincubation is in a chamber that previously held said high pHdesulphonation buffer (e.g., chamber 10).

Embodiment 59

The method according to any one of embodiments 1-58, wherein after theincubation with bisulfite ions, a temperature controlled channel orchamber is washed with a buffer to remove the residual bisulfite andneutralize pH.

Embodiment 60

The method according to any one of embodiments 1-59, wherein highresolution melting analysis (HRM) on said bisulfite-converted nucleicacid is performed to determine the methylation of said nucleic acid.

Embodiment 61

The method according to any one of embodiments 1-60, wherein nucleicacid sequencing of said bisulfite-converted nucleic acid is performed todetermine the methylation of said nucleic acid.

Embodiment 62

The method according to any one of embodiments 1-60, wherein methylationspecific PCR is performed to determine methylation of target nucleicacid sequences.

Embodiment 63

The method of embodiment 62, wherein said methylation specific PCR (MSP)is performed using primers specific for methylated sequences and/orprimers specific for unmethylated sequences.

Embodiment 64

The method of embodiment 62, wherein said methylation specific PCRcomprises a MethyLight protocol.

Embodiment 65

The method of embodiment 62, wherein TaqMan PCR reactions are performedwith primers specific for bisulfite-converted methylated and/orunmethylated sequences.

Embodiment 66

The method according to any one of embodiments 62-65, wherein said MSPutilizes one or more fluorescent probes that are markers for amplifiedmethylated sequences and/or one or more fluorescent probes that aremarkers for amplified unmethylated sequences.

Embodiment 67

The method of embodiment 66, wherein said fluorescent probes comprise afluorescent reporter dye and a quencher dye where the probe provides asignal upon cleavage by 5′ to 3′ nuclease activity of Taq DNApolymerase.

Embodiment 68

The method according to any one of embodiments 66-67, wherein amethylation signal is determined by the combined signal for a pluralityof probes each specific to a different methylated region in an amplifiedregion of interest.

Embodiment 69

The method according to any one of embodiments 66-67, wherein amethylation signal is determined by a plurality of probes specific forthe same methylated region in an amplified region of interest.

Embodiment 70

The method according to any one of embodiments 66-67, wherein saidplurality of probes comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more probes.

Embodiment 71

The method according to any one of embodiments 66-67, wherein amethylation signal is determined by a single probe in the amplifiedregion of interest.

Embodiment 72

The method according to any one of embodiments 66-71, wherein saidprobes are run in simplex or multiplex.

Embodiment 73

The method according to any one of embodiments 66-71, wherein saidprobes are run in a multiplex format.

Embodiment 74

The method according to any one of embodiments 66-73, wherein saidprobes are run as a nested PCR reaction.

Embodiment 75

The method according to any one of embodiments 66-74, wherein said PCRreaction comprises a bisulfite contamination control probe that thatundergoes bisulfite-mediated cleavage during PCR if bisulfite is presentin the reaction.

Embodiment 76

The method according to any one of embodiments 1-75, wherein PCR isperformed for one or more mutated genes.

Embodiment 77

The method according to any one of embodiments 1-76, wherein PCR isperformed for unconverted DNA as a control.

Embodiment 78

The method according to any one of embodiments 1-77, wherein PCR isperformed for converted DNA as a control.

Embodiment 79

The method of embodiment 77, wherein PCR is performed for unconvertedDNA where the unconverted DNA is a target for said method.

Embodiment 80

The method according to any one of embodiments 1-79, wherein a bisulfitereaction and a PCR reaction, or a desulphonation reaction and a PCRreaction, or a bisulfite reaction, a desulphonation reaction and a PCRreaction are all performed in the same reaction tube or chamber.

Embodiment 81

The method according to any one of embodiments 1-80, wherein saidcontacting a biological sample comprising a nucleic acid to a firstmatrix material comprises contacting a sample containing RNA to saidfirst matrix material, where said matrix material binds said RNA therebypurifies the RNA.

Embodiment 82

The method of embodiment 81, wherein said method comprises eluting saidRNA from said matrix material substantially independently of the DNA.

Embodiment 83

The method of embodiment 82, wherein the RNA is eluted from said firstmatrix material using a Tris buffered elution.

Embodiment 84

The method according to any one of embodiments 81-83, wherein said RNAis eluted and stored in a chamber.

Embodiment 85

The method according to any one of embodiments 81-84, wherein reversetranscription (RT) is performed on said RNA and qRT-PCR is performed todetermine the level of target RNA sequences.

Embodiment 86

The method according to any one of embodiments 82-85, where the RNAfraction is used to elute the bisulfite converted nucleic acid from saidsecond matrix material and mix with the bisulfite-converted DNA, or ismixed with eluted bisulfite-converted DNA.

Embodiment 87

The method of embodiment 86, wherein RT is performed on said RNA priorto, or after, combination with the bisulfite-converted DNA.

Embodiment 88

The method according to any one of embodiments 86-87, wherein qRT-PCR isperformed for RT RNA in the mixture to determine the level of target RNAsequences and methylation specific PCR is performed on the mixture todetermine methylation of target DNA sequences.

Embodiment 89

The method according to any one of embodiments 1-88, where methylationis determined for a promoter region of a gene selected from the groupconsisting of MGMT. RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2,TM6SF1, and AKR1B1.

Embodiment 90

The method according to any one of embodiments 81-89, wherein theexpression level of RNA is determined for a methyltransferase.

Embodiment 91

The method of embodiment 90, wherein the expression level of RNA isdetermined for a methyltransferase selected from the group consisting ofDNMT1, DNMT2, DNMT3 A, DNMT3B, and TNMT3L.

Embodiment 92

A cartridge for determining the methylation state of a nucleic acid,said cartridge comprising: a column comprising a first matrix material,a sample receiving chamber, a temperature controlled channel or chamber,a plurality of chambers containing reagents and/or buffers, and when inuse at least one of said chambers contains a bisulfite reagent, and atleast one of said chambers contains a desulphonation/elution buffer, andwherein said cartridge optionally comprises a second column comprisingsaid second matrix material.

Embodiment 93

The cartridge of embodiment 92, wherein said cartridge, when in use,comprises a chamber containing a reagent comprising guanidiniumthiocyanate ethanol (GTC-EtOH).

Embodiment 94

The cartridge according to any one of embodiments 92-93, wherein saidsecond column is absent.

Embodiment 95

The cartridge according to any one of embodiments 92-93, wherein saidsecond column is present.

Embodiment 96

The cartridge according to any one of embodiments 92-95, wherein saidtemperature controlled channel or chamber is a thermocycling channel orchamber.

Embodiment 97

The cartridge according to any one of embodiments 92-96, wherein saidcartridge further comprises a second heating channel or chamber.

Embodiment 98

The cartridge according to any one of embodiment 92-97, wherein saidbisulfite reagent comprises a compound selected from the groupconsisting of ammonium bisulfite, sodium metabisulfite, potassiumbisulfite, cesium bisulfite, and DABSO.

Embodiment 99

The cartridge of embodiment 98, wherein said bisulfite reagent comprisesammonium bisulfite.

Embodiment 100

The cartridge according to any one of embodiments 92-99, wherein saidbisulfite is provided in a reagent mix comprising scavengers to preventsulfite oxidation and/or catalysts.

Embodiment 101

The cartridge of embodiment 100, wherein said bisulfite is provided in areagent mix comprising scavengers selected from the group consisting ofTrolox and hydroquinone.

Embodiment 102

The cartridge according to any one of embodiments 100-101, wherein saidbisulfite is provided in a reagent mix comprising polyamines ascatalysts.

Embodiment 103

The cartridge according to any one of embodiments 92-102, wherein saidfirst matrix material and/or said second matrix material, when present,comprises a material is selected from the group consisting of glass orsilica, an ion exchange resin, and hydroxyapatite.

Embodiment 104

The cartridge according to any one of embodiments 92-103, wherein saidcartridge comprises one or more chambers containing one or more reagentsselected from the group consisting of methylation specific PCR primers,methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer.

Embodiment 105

The cartridge of embodiment 104, wherein said cartridge contains atleast two chambers containing one or more reagents selected from thegroup consisting of methylation specific PCR primers, methylationspecific PCR probes, PCR enzyme(s), and PCR reaction buffer.

Embodiment 106

The cartridge according to any one of embodiments 92-105, wherein saidcartridge contains at least one chamber containing primers and probesfor detection of methylation of a forward strand of a converted DNA.

Embodiment 107

The cartridge according to any one of embodiments 92-106, wherein saidcartridge contains at least one chamber containing primers and probesfor detection of methylation of a reverse strand of a converted DNA.

Embodiment 108

The cartridge according to any of embodiments 104-107, wherein said PCRprimers, and/or probes, and/or enzymes are provided as beads.

Embodiment 109

The cartridge according to any one of embodiments 92-108, wherein saidsample receiving chamber, said column(s), said plurality of chambers,and said temperature-controlled heating channel or chamber, areselectively in fluid communication.

Embodiment 110

The cartridge of embodiment 109, wherein said sample receiving chamber,said column(s), said plurality of chambers, and said temperaturecontrolled channel or chamber, are selectively in fluid communication bymicrofluidic channels and valves.

Embodiment 111

The cartridge of embodiment 109, wherein said sample receiving chamber,said column(s), said plurality of chambers, and said temperaturecontrolled channel or chamber or a port into said temperature controlledchannel or chamber, are disposed around a central valve and selectivelyin fluid communication with a channel in said central valve, whereinsaid central valve is configured to accommodate a plunger that iscapable of drawing fluid into or out of a chamber in fluid communicationwith said central valve.

Embodiment 112

The cartridge according to any one of embodiments 92-111, wherein saidcartridge is configured so that, when in use, said cartridge comprises:

a first chamber containing a sample;

a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH)solution;

a third chamber containing a bisulfite reagent;

a fourth chamber containing a buffer;

a fifth chamber containing a rinse solution; and

a sixth chamber containing an elution/desulphonation reagent.

Embodiment 113

The cartridge of embodiment 112, wherein said first chamber containssaid sample in a GTC-EtOH-Tween extraction/precipitation reagent.

Embodiment 114

The cartridge according to any one of embodiments 92-113, wherein thecartridge is configured for the bisulfite reagent to be added to thecartridge at or near the time the sample is placed in the cartridge.

Embodiment 115

The cartridge according to any one of embodiments 92-113, wherein thebisulfite reagent is provided as a component of the cartridge.

Embodiment 116

The cartridge according to any one of embodiments 92-115, wherein thecartridge is configured for addition of GTC-ETOH-Tween buffer at or nearthe time the sample is placed into the cartridge.

Embodiment 117

The cartridge according to any one of embodiments 92-115, wherein theGTC-ETOH-Tween buffer is provided as a component of the cartridge.

Embodiment 118

The cartridge according to any one of embodiments 92-117, wherein saidcartridge comprises a seventh chamber containing PCR primers and/orprobes and/or PCR enzymes.

Embodiment 119

The cartridge according to any one of embodiments 92-118, wherein saidcartridge comprises an eighth chamber also containing PCR primers and/orprobes and/or PCR enzymes.

Embodiment 120

The cartridge according to any one of embodiments 92-119, wherein saidcartridge comprises one or more chambers containing primers specific forbisulfite-converted methylated and/or unmethylated sequences.

Embodiment 121

The cartridge according to any one of embodiments 92-120, wherein saidcartridge comprises one or more chambers containing reagents for TaqManPCR reactions.

Embodiment 122

The cartridge according to any one of embodiments 92-121, wherein saidcartridge comprises one or more chambers containing one or morefluorescent probes that are markers for amplified methylated sequencesand/or one or more fluorescent probes that are markers for amplifiedunmethylated sequences.

Embodiment 123

The cartridge of embodiment 122, wherein said probes comprise afluorescent reporter dye and a quencher dye, where the probes provides asignal upon cleavage by the 5′ to 3′ nuclease activity of Taq DNApolymerase.

Embodiment 124

The cartridge according to any one of embodiments 122-123, wherein saidcartridge comprises a plurality of probes each specific to a differentmethylated region in an amplified region of interest.

Embodiment 125

The cartridge according to any one of embodiments 122-123, wherein saidcartridge comprises a single probe specific to a methylated region in anamplified region of interest.

Embodiment 126

The cartridge according to any one of embodiments 122-123, wherein saidcartridge comprises a plurality of probes each specific to the samemethylated region in an amplified region of interest.

Embodiment 127

The cartridge according to any one of embodiments 92-126, wherein saidcartridge contains primers and/or probes to determine methylation of apromoter region of a gene selected from the group consisting of MGMT,RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, and AKR1B1.

Embodiment 128

The cartridge according to any one of embodiments 92-126, wherein saidcartridge contains one or more primers shown in Tables 5, 9, or 10,and/or one or more probes shown in Tables 5, 9, or 10.

Embodiment 129

The cartridge of embodiment 128, wherein said cartridge contains thefollowing probes and primers for determining methylation of MGMT using anested PCR reaction:

an external forward primer (248b) comprising the nucleotide sequenceGTTTT(T*)AGAAYG(T*)TTTGYGTTT (SEQ ID NO:263);

an external reverse primer (249b) comprising the nucleotide sequence:AAAAAAC(T*)CCRCACTCTTCC (SEQ ID NO:265);

an internal forward primer (250) comprising the nucleotide sequenceTTTCGACGTTCGTAGGTTTTCGC (SEQ ID NO:266);

an internal reverse primer (251) comprising the nucleotide sequenceGCACTCTTCCGAAAACGAAACG (SEQ ID NO:267); and

a probe (252a) comprising the nucleotide sequencefluor-CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker (SEQ ID NO: 268).

Embodiment 130

The cartridge according to any one of embodiments 128-129, wherein saidcartridge contains the following probes and primers for determiningmethylation of ACTB (e.g., as a control) using a nested PCR reaction:

an external forward primer (102) comprising the nucleotide sequence:GTGATGGAGGAGGTTTAGTAAGTT (SEQ ID NO:103);

an external reverse primer (103) comprising the nucleotide sequence:CCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO:104);

an internal forward primer (148) comprising the nucleotide sequence:GGTTTAGTAAGTTTTTTGGATTGTG (SEQ ID NO:149);

an internal reverse primer (149) comprising the nucleotide sequence:CCTTAAAAATTACAAAAACCACAAC (SEQ ID NO:150); and

a probe (178) comprising the nucleotide sequence:fluor-CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker (SEQ ID NO:179).

Embodiment 131

The cartridge according to any one of embodiments 92-130, wherein thecartridge is configured for determination of the expression level of RNAfor a methyltransferase.

Embodiment 132

The cartridge of embodiment 131, wherein said methyltransferases isselected from the group consisting of DNMT1, DNMT2, DNMT3A, DNMT3B, andTNMT3L.

Embodiment 133

A system for determining the methylation of a nucleic acid in abiological sample, said system comprising: an enclosure configured tocontain one or more sample processing modules, each sample processingmodule configured to hold a removable cartridge according to any one ofembodiments 92-132; where said system is configured to operate thesample processing modules to perform sample processing to determinemethylation of one or more target nucleic acids and optionally todetermine the level of one or more target DNA sequences within acorresponding removable sample cartridge, wherein said processing on asample within the corresponding removable sample cartridge performs amethod according to any one of embodiments 1-91.

Embodiment 134

The system of embodiment 133, wherein said system is configured tocontain one sample processing module.

Embodiment 135

The system of embodiment 133, wherein said system is configured tocontain at least two sample processing modules, or at least 4 sampleprocessing modules, or at least 8 sample processing modules, or at least12 sample processing modules, or at least 16 sample processing modules,or at least 20 sample processing modules, or at least 24 sampleprocessing modules, or at least 28 sample processing modules, or atleast 32 sample processing modules, or at least 64 sample processingmodules, or at least 128 sample processing modules.

Embodiment 136

The system according to any one of embodiments 133-135, wherein saidmodules comprise one or more heating plates to heat a temperaturecontrolled chamber or channel in said cartridge.

Embodiment 137

The system according to any one of embodiments 133-136, wherein saidmodules comprise a fan configured to cool a temperature controlledchannel or chamber in said cartridge.

Embodiment 138

The system according to any one of embodiments 133-137, wherein saidmodules comprise circuitry to pass information (e.g., opticalinformation) to a computer for analysis.

Embodiment 139

The system according to any one of embodiments 133-138, wherein saidmodules comprise optical blocks to provide excitation and/or detectionof one or more optical signals produced by reactions in said cartridge.

Embodiment 140

The system according to any one of embodiments 133-139, wherein saidsystem is configured to operate said cartridge to perform a methodaccording to any one of embodiments 1-91.

Embodiment 141

The system according to any one of embodiments 133-139, wherein saidsystem is configured to operate said cartridge to: bind a sample to acolumn; elute DNA from the column and combine said DNA with a conversionreagent; heat the DNA/conversion reagent solution in a reaction chamberor tube to produce converted DNA; bind the converted DNA to a column;desulphonate and elute the DNA from the column; and perform PCR on theeluted desulphonated DNA in a reaction chamber or tube.

Embodiment 142

The system of embodiment 141, wherein said PCR is performed in the samereaction chamber or tube where the DNA/conversion reagent solution waspreviously heated.

Embodiment 143

A cartridge for sample preparation, said cartridge comprising: a channelor chamber comprising an affinity matrix that binds DNA, a plurality ofchambers disposed around a central valve assembly and selectively influid communication with said central valve assembly where said centralvalve assembly is configured to accommodate a plunger that is capable ofdrawing fluid into or out of a chamber in fluid communication with saidcentral valve wherein said plurality of chambers comprises: a chamberconfigured to receive up to about 5 ml or up to about 4 ml of samplesolution; a chamber containing PEG; a chamber containing GTC-EtOH; achamber containing an alkaline solution; and a chamber containing abuffer.

Embodiment 144

The cartridge of embodiment 143, wherein said plurality of chambersfurther comprises a chamber containing a bisulfite reagent.

Embodiment 145

The cartridge according to any one of embodiments 143-144, wherein saidplurality of chambers comprises a chamber containing a GTC-ethanol washsolution.

Embodiment 146

The cartridge of embodiment 145, wherein said GTC-ethanol wash solutioncomprises 1.25M guanidinium thiocyanate, 25 mM Tris pH 7.0, and 50%ethanol.

Embodiment 147

The cartridge according to any one of embodiments 143-146, wherein saidPEG comprises PEG200.

Embodiment 148

The cartridge according to any one of embodiments 143-147, wherein saidalkaline solution comprises KOH.

Embodiment 149

The cartridge according to any one of embodiments 143-148, wherein saidbuffer comprises Tris.

Embodiment 150

The cartridge according to any one of embodiments 143-149. wherein saidplurality of chambers comprises a chamber containing beads comprisingone or more PCR primers and/or probes.

Embodiment 151

The cartridge according to any one of embodiments 143-150, wherein saidchamber containing PEG contains about 1 ml of PEG.

Embodiment 152

The cartridge according to any one of embodiments 143-151, wherein saidchamber containing an alkaline solution contains about 500 μL ofsolution.

Embodiment 153

The cartridge according to any one of embodiments 143-152, wherein saidchamber containing GTC-EtOH contains about 2 ml GTC-EtOH.

Embodiment 154

The cartridge according to any one of embodiments 143-153, wherein saidchamber containing a buffer contains about 2 mL of buffer.

Embodiment 155

A high volume sample preparation (HVSP), said cartridge comprising: achannel or chamber comprising an affinity matrix that binds DNA, aplurality of chambers disposed around a central valve assembly andselectively in fluid communication with said central valve assemblywhere said central valve assembly is configured to accommodate a plungerthat is capable of drawing fluid into or out of a chamber in fluidcommunication with said central valve wherein said plurality of chamberscomprises: at least two different chambers each configured to receive upto about 4.5 ml of sample solution; a chamber containing PEG; a chambercontaining an alkaline solution; and a chamber containing a buffer.

Embodiment 156

The cartridge of embodiment 155, wherein said plurality of chamberscomprises at least three different chambers each configured to receiveup to 4 ml of sample solution.

Embodiment 157

The cartridge according to any one of embodiments 155-156, wherein saidPEG comprises PEG200.

Embodiment 158

The cartridge according to any one of embodiments 155-157, wherein saidbasic solution comprises KOH.

Embodiment 159

The cartridge according to any one of embodiments 155-158, wherein saidbuffer comprises Tris.

Embodiment 160

The cartridge according to any one of embodiments 155-159, wherein saidplurality of chambers comprises a chamber containing a wash solution.

Embodiment 161

The cartridge of embodiment 160, wherein said wash solution comprise1.25M guanidinium thiocyanate, 25 mM Tris pH 7.0, and 50% ethanol.

Embodiment 162

The cartridge according to any one of embodiments 155-161, wherein saidcartridge comprises a chamber configured for removal of a processedsample.

Embodiment 163

The cartridge according to any one of embodiments 155-162, wherein saidsample chambers, when in use contain sample solution, GTC andisopropanol.

Embodiment 164

The cartridge of embodiment 163, wherein said sample chambers, when inuse contain sample solution, GTC and isopropanol in substantially equalvolumes.

Embodiment 165

The cartridge according to any one of embodiments 155-164 wherein saidcartridge, when in use, comprises 4 ml of sample solution disposed ineach of said chambers configured to receive a sample.

Embodiment 166

The cartridge according to any one of embodiments 155-165, wherein saidcartridge provides DNA or RNA recovery that is substantially linear withrespect to the sample volume between 0.5 ml and about 4 ml of sample.

Embodiment 167

The cartridge according to any one of embodiments 155-166, wherein saidcartridge contains or is configured to receive a conversion reagent.

Embodiment 168

The cartridge of embodiment 167, wherein said cartridge, when in use,performs a bisulfite conversion of DNA.

Embodiment 169

A lysis solution for preparation of a DNA sample from serum or plasma,said lysis solution comprising: GTC, a buffer, a detergent, andoptionally an anti-foaming agent.

Embodiment 170

The lysis solution of embodiment 169, wherein said lysis solution forserum or plasma comprises GTC, Tris pH 7.0, Tween 20, and antifoam SE15.

Embodiment 171

The lysis solution of embodiment 170, wherein said lysis solution forserum or plasma comprises about 4.5M GTC, about 45 mM Tris pH 7.0, about1% Tween20, and about 0.01% Antifoam SE15.

Embodiment 172

A lysis solution for preparation of a DNA sample from an FFPE sample.

Embodiment 173

The lysis solution of embodiment 172, wherein said lysis solution forFFPE samples comprises a buffer, a detergent, NaCl, MgCl₂, a chelatingagent, antifoam SE15, and sodium azide.

Embodiment 174

The lysis solution of embodiment 173, wherein said lysis solution forFFPE samples comprises about 1% Tween20, about 400 mM NaCl, about 25 mMEDTA, about 10 mM MgCl₂, about 50 mM HEPES pH 7.2, about 0.01% antifoamSE15, and about 0.01% sodium azide.

Embodiment 175

A kit for the determination of DNA methylation, said kit comprising: acontainer containing a cartridge for determining the methylation stateof a nucleic acid according to any one of embodiments 92-136.

Embodiment 176

The kit of embodiment 175, wherein said kit further comprises acontainer containing a lysis solution.

Embodiment 177

The kit of embodiment 176, wherein said lysis solution is a lysissolution for serum or plasma according to any one of embodiments169-171.

Embodiment 178

The kit of embodiment 176, wherein said lysis solution is a lysissolution for an FFPE sample according to any one of embodiments 172-174.

Embodiment 179

The kit according to any one of embodiments 175-178, wherein said kitcomprises a container containing proteinase K.

Embodiment 180

The kit according to any one of embodiments 175-179, wherein said kitcomprises a conversion reagent in said cartridge or in a containerseparate from the cartridge.

Embodiment 181

The kit of embodiment 180, wherein said kit comprises said conversionreagent in a container separate from the cartridge.

Embodiment 182

The kit of embodiment 180, wherein said kit comprises said conversionreagent is provided in a chamber of the cartridge.

Embodiment 183

The according to any one of embodiments 180-182, wherein said conversionreagent comprises a compound selected from the group consisting ofsodium metabisulfite, potassium bisulfite, cesium bisulfite, ammoniumbisulfite, and DABSO.

Embodiment 184

The kit of embodiment 183, wherein said conversion reagent comprisesammonium bisulfite.

Embodiment 185

The kit according to any one of embodiments 175-184, wherein said kitcomprises a container containing a sample processing reagent.

Embodiment 186

The kit of embodiment 185, wherein said sample processing reagentcomprises guanidium thiocyanate.

Embodiment 187

The kit according to any one of embodiments 185-186, wherein said sampleprocessing reagent comprise ethanol.

Embodiment 188

The kit according to any one of embodiments 175-187, wherein said kitcomprises a container containing a cartridge for sample preparationaccording to any one of embodiments 155-166.

Embodiment 189

The kit according to any one of embodiments 175-188, wherein said kitcontains instructional materials teaching the use of said cartridge forthe determination of DNA methylation.

Embodiment 190

A cartridge for the detection of methylation markers of a cancer, saidcartridge comprising: a plurality of chambers and a thermocyclingchannel or chamber, wherein said plurality of chambers and a port intosaid thermocycling channel or chamber are disposed around a centralvalve assembly and selectively in fluid communication with said centralvalve assembly where said central valve assembly is configured toaccommodate a plunger that is capable of drawing fluid into or out of achamber or port in fluid communication with said central valve whereinsaid plurality of chambers comprises: a sample receiving chamber; achamber containing or configured to receive a bisulfite reagent; achamber containing a wash solution; a chamber containing a Tris buffer;a chamber containing an alkaline solution comprising KOH; a chambercontaining beads that provide a PCR master mix; and a chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters whose methylation state is a marker for a cancer.

Embodiment 191

The cartridge of embodiment 190, wherein said plurality of chamberscomprises a chamber disposed to receive waste solutions.

Embodiment 192

The cartridge according to any of embodiments 190-191, wherein saidbisulfite reagent comprises a compound selected from the groupconsisting of sodium metabisulfite, potassium bisulfite, cesiumbisulfite, ammonium bisulfite, and DABSO.

Embodiment 193

The cartridge of embodiment 192, wherein said bisulfite reagentcomprises ammonium bisulfite.

Embodiment 194

The cartridge according to any of embodiments 190-193, wherein said washsolution comprises 1.25M GTC, 25 mM Tris pH 7.0, and 50% ethanol.

Embodiment 195

The cartridge according to any of embodiments 190-194, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for a cancer selected fromthe group consisting of breast cancer, pancreatic cancer, prostatecancer, brain cancer, and lung cancer.

Embodiment 196

The cartridge of embodiment 195, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes for anested PCR reaction.

Embodiment 197

The cartridge of embodiment 196, wherein said nested PCR comprises afirst PCR reaction specific for converted DNA and a second PCR reactionspecific for methylated CpGs.

Embodiment 198

The cartridge according to any one of embodiments 190-197, wherein saidchamber containing beads that provide PCR primers and probes chambercontains beads that provide PCR primers and probes to detect methylationof a forward strand of converted DNA.

Embodiment 199

The cartridge according to any one of embodiments 190-198, wherein saidchamber containing beads that provide PCR primers and probes chambercontains beads that provide PCR primers and probes to detect methylationof a reverse strand of converted DNA.

Embodiment 200

The cartridge according to any of embodiments 190-199, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoters of one ormore genes selected from the group consisting of RASSF1A, AKR1B1, HOXB4,HIST1H3C, RASGRF2, TM6SF1, BRCA1, BNC1, ADAMTS1, CDO1, SOX17, TAC1,HOXA7, and MGMT.

Embodiment 201

The cartridge according to any of embodiments 190-200, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for pancreatic cancer.

Embodiment 202

The cartridge of embodiment 201, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes todetect methylation of the promoters of ADAMTS1, and/or BNC1.

Embodiment 203

The cartridge of embodiment 202, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes todetect methylation of the promoter of ADAMTS1.

Embodiment 204

The cartridge according to any one of embodiments 202-203, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of BNC1.

Embodiment 205

The cartridge of embodiment 202, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide one or more PCR primersand/or probes for ADAMTS1 and/or BNC1 shown in Tables 5, or 10.

Embodiment 206

The cartridge according to any of embodiments 190-200, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for breast cancer.

Embodiment 207

The cartridge of embodiment 206, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes todetect methylation of the promoters of one, two, three, four, five, orall genes selected from the group consisting of BRCA1, RASSF1A, AKR1B1,HOXB4, HIST1H3C, RASGRF2, and TM6SF1.

Embodiment 208

The cartridge of embodiment 207, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes todetect methylation of the promoter of BRCA1.

Embodiment 209

The cartridge according to any one of embodiments 207-208, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of RASSF1A.

Embodiment 210

The cartridge according to any one of embodiments 207-209, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of AKRIB1.

Embodiment 211

The cartridge according to any one of embodiments 207-210, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of HOXB4.

Embodiment 212

The cartridge according to any one of embodiments 207-211, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter ofHIST1H3C.

Embodiment 213

The cartridge according to any one of embodiments 207-212, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of RASGRF2.

Embodiment 214

The cartridge according to any one of embodiments 207-213, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of TM6SF1.

Embodiment 215

The cartridge according to any one of embodiments 207-214, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that provideone or more PCR primers and/or one or more PCR probes shown in Tables 5,or 9.

Embodiment 216

The cartridge of embodiment 206, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes todetect methylation of the promoters of BRCA1.

Embodiment 217

The cartridge according to any of embodiments 190-200, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for lung cancer.

Embodiment 218

The cartridge of embodiment 217, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes todetect methylation of the promoters of one, two, three, or all genesselected from the group consisting of CDO1, SOX17, TAC1, and HOXA7.

Embodiment 219

The cartridge of embodiment 218, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes todetect methylation of the promoter of CDO1.

Embodiment 220

The cartridge according to any one of embodiments 218-219, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of SOX17.

Embodiment 221

The cartridge according to any one of embodiments 218-220, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of TAC1.

Embodiment 222

The cartridge according to any one of embodiments 218-221, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of the promoter of HOXA7.

Embodiment 223

The cartridge according to any of embodiments 190-200, wherein saidchamber containing beads that provide PCR primers and probes to detectmethylation of one or more gene promoters comprises beads that providePCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for brain cancer.

Embodiment 224

The cartridge of embodiment 223, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide PCR primers and probes todetect methylation of the promoter of MGMT.

Embodiment 225

The cartridge of embodiment 224, wherein said chamber containing beadsthat provide PCR primers and probes to detect methylation of one or moregene promoters comprises beads that provide one or more PCR primersand/or probes for MGMT shown in Tables 5, or 10.

Embodiment 226

The cartridge of embodiment 225, wherein said cartridge contains thefollowing probes and primers for determining methylation of MGMT using anested PCR reaction:

an external forward primer (248b) comprising the nucleotide sequenceGTTTT(T*)AGAAYG(T*)TTTGYGTTT (SEQ ID NO:263);

an external reverse primer (249b) comprising the nucleotide sequenceAAAAAAC(T*)CCRCACTCTTCC (SEQ ID NO:265);

an internal forward primer (250) comprising the nucleotide sequenceTTTCGACGTTCGTAGGTTTTCGC (SEQ ID NO:266);

an internal reverse primer (251) comprising the nucleotide sequenceGCACTCTTCCGAAAACGAAACG (SEQ ID NO:267); and

a probe (252a) comprising the nucleotide sequencefluor-CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker (SEQ ID NO: 268).

Embodiment 227

The cartridge according to any one of embodiments 225-226, wherein saidcartridge contains the following probes and primers for determiningmethylation of ACTB (e.g., as a control) using a nested PCR reaction:

an external forward primer (102) comprising the nucleotide sequenceGTGATGGAGGAGGTTTAGTAAGTT (SEQ ID NO:103);

an external reverse primer (103) comprising the nucleotide sequenceCCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO:104);

an internal forward primer (148) comprising the nucleotide sequenceGGTTTAGTAAGTTTTTTGGATTGTG (SEQ ID NO:149);

an internal reverse primer (149) comprising the nucleotide sequenceCCTTAAAAATTACAAAAACCACAAC (SEQ ID NO:150); and

a probe (178) comprising the nucleotide sequencefluor-CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker (SEQ ID NO:179).

Embodiment 228

A method of preparing a sample of cfDNA from serum or plasma, saidmethod comprising:

combining a proteinase K treated sample of serum or plasma with a lysissolution according to any one of embodiments 169-171, and an alcohol toform a sample solution;

loading said sample solution into a sample receiving chamber in acartridge according to any one of embodiments 143-154, or into a samplereceiving chamber in a cartridge according to any one of embodiments155-168; and

operating said cartridge to bind DNA in said sample to said affinitymatrix and then to wash and release said DNA from said matrix.

Embodiment 229

The method of embodiment 228, wherein said combining a proteinase Ktreated sample of serum or plasma comprises combining said sample, lysissolution and alcohol in proportions corresponding to about 1.3 mlproteinase K treated serum or plasma, 2.2 mL lysis solution; and about1.5 ml alcohol.

Embodiment 230

The method according to any one of embodiments 228-229, wherein saidalcohol comprises isopropanol.

Embodiment 231

The method according to any one of embodiments 228-230, wherein saidsample comprises serum.

Embodiment 232

The method according to any one of embodiments 228-231, wherein saidsample comprises plasma.

Embodiment 233

The method according to any one of embodiments 228-232, wherein saidsample comprises serum.

Embodiment 234

The method according to any one of embodiments 228-233, whereinoperating said cartridge comprises introducing said cartridge into asample processing module in a system according to any one of embodiments133-139.

Embodiment 235

The method according to any one of embodiments 228-234, wherein saidmethod further comprises operating said cartridge to convert said DNAfor methylation detection.

Embodiment 236

The method according to any one of embodiments 228-235, wherein saidmethod further comprises operating said cartridge to perform one or morePCR reactions using said DNA or converted DNA a template.

Embodiment 237

The method according to any one of embodiments 228-234, wherein saidloading comprises loading said sample solution into one or more samplereceiving chambers in a cartridge according to any one of embodiments155-165.

Embodiment 238

The method of embodiment 237, wherein said method further comprisestransferring the released DNA to a second cartridge for methylationdetection and/or PCR.

Embodiment 239

The method of embodiment 238, wherein said second cartridge is acartridge according to any one of embodiments 92-132.

Embodiment 240

The method according to any one of embodiments 238-239, wherein saidmethod further comprises operating said second cartridge to convert saidDNA for methylation detection.

Embodiment 241

The method according to any one of embodiments 238-240, wherein saidmethod further comprises operating said second cartridge to perform oneor more PCR reactions using said DNA or converted DNA as a template.

Embodiment 242

The method according to any one of embodiments 238-241, wherein saidoperating said second cartridge comprises introducing said secondcartridge into a sample processing module in a system according to anyone of embodiments 133-139.

Embodiment 243

A method of preparing a DNA from an FFPE sample, said method comprising:

combining a formalin-fixed paraffin embedded sample with a lysissolution according to any one of embodiments 172-174;

heating said lysis solution containing said sample; adding an alcohol tosaid sample to form a sample solution; loading said sample solution intoa sample receiving chamber in a cartridge according to any one ofembodiments 143-154, or into a sample receiving chamber in a cartridgeaccording to any one of embodiments 155-168; and

operating said cartridge to bind DNA in said sample to said affinitymatrix and then to wash and release said DNA from said matrix.

Embodiment 244

The method of embodiment 243, wherein said heating comprises addingproteinase K to said sample and heating said sample.

Embodiment 245

The method of embodiment 244, wherein said heating comprises addingabout 50 μL proteinase K to about 1.2 mL of FFPE lysis solutioncontaining a FFPE sample.

Embodiment 246

The method according to any one of embodiments 243-245, wherein saidheating comprises heating said lysis solution to a temperature rangingfrom about 50° C. to about 60° C.

Embodiment 247

The method of embodiment 246, wherein said heating comprises heatingsaid lysis solution to a temperature of about 56° C.

Embodiment 248

The method according to any one of embodiments 243-247, wherein saidheating is for a period of time ranging up to about 4 hours, or up toabout 5 hours, or up to about 6 hours.

Embodiment 249

The method of embodiment 248, wherein said heating is for about 4 hours.

Embodiment 250

The method according to any one of embodiments 243-249, wherein saidalcohol comprises ethanol.

Embodiment 251

The method according to any one of embodiments 243-250, wherein saidmethod comprises adding alcohol to said lysis solution in a volume ratioof about 1:1 lysis solution:alcohol.

Embodiment 252

The method according to any one of embodiments 243-251, whereinoperating said cartridge comprises introducing said cartridge into asample processing module in a system according to any one of embodiments133-139.

Embodiment 253

The method according to any one of embodiments 243-252, wherein saidmethod further comprises operating said cartridge to convert said DNAfor methylation detection.

Embodiment 254

The method according to any one of embodiments 243-253, wherein saidmethod further comprises operating said cartridge to perform one or morePCR reactions using said DNA or converted DNA as a template.

Embodiment 255

The method according to any one of embodiments 243-251, wherein saidloading comprise loading said sample solution into one or more samplereceiving chambers in a cartridge according to any one of embodiments155-165.

Embodiment 256

The method of embodiment 255, wherein said method further comprisestransferring the released DNA to a second cartridge for methylationdetection and/or PCR.

Embodiment 257

The method of embodiment 256, wherein said second cartridge is acartridge according to any one of embodiments 92-132.

Embodiment 258

The method according to any one of embodiments 256-257, wherein saidmethod further comprises operating said second cartridge to convert saidDNA for methylation detection.

Embodiment 259

The method according to any one of embodiments 256-258, wherein saidmethod further comprises operating said second cartridge to perform oneor more PCR reactions using said DNA or converted DNA as a template.

Embodiment 260

The method according to any one of embodiments 256-259, wherein saidoperating said second cartridge comprises introducing said secondcartridge into a sample processing module in a system according to anyone of embodiments 133-139.

Embodiment 261

A method of detecting a cancer, and/or staging a cancer, and/ordetecting the predisposition to a cancer in a subject, said methodcomprising:

providing a biological sample from said subject, wherein said biologicalsample comprises a DNA;

utilizing a cartridge according to any one of claims 190-225 to detectmethylation of one or more gene promoters in said DNA whose methylationstate is a marker for a cancer, where an increase in methylation of saidone or more gene promoters is indicative of the presence of a cancer ora predisposition to a cancer or a stage of a cancer or precancer.

Embodiment 262

The method of embodiment 261, wherein said subject is a human.

Embodiment 263

The method according to any one of embodiments 261-262, wherein saidcancer is a cancer selected from the group consisting of breast cancer,pancreatic cancer, prostate cancer, brain cancer, a lung cancer, a Bcell lymphoma, a bronchus cancer, a colorectal cancer, a stomach cancer,an ovarian cancer, a urinary bladder cancer, a brain or central nervoussystem cancer, a peripheral nervous system cancer, an esophageal cancer,a cervical cancer, a melanoma, a uterine or endometrial cancer, a cancerof the oral cavity or pharynx, a liver cancer, a kidney cancer, abiliary tract cancer, a small bowel or appendix cancer, a salivary glandcancer, a thyroid gland cancer, a adrenal gland cancer, an osteosarcoma,a chondrosarcoma, a liposarcoma, a testes cancer, and a malignantfibrous histiocytoma.

Embodiment 264

The method according to any one of embodiments 261-262, wherein saidcancer is a cancer selected from the group consisting of breast cancer,pancreatic cancer, prostate cancer, brain cancer, a lung cancer.

Embodiment 265

The method according to any one of embodiments 261-264, wherein saidsample comprise a sample from serum or plasma.

Embodiment 266

The method according to any one of embodiments 261-264, wherein saidsample comprise an FFPE sample.

Embodiment 267

The method according to any one of embodiments 261-266, wherein said oneor more gene promoters comprise the promoters of one or more genesselected from the group consisting of RASSF1A, AKR1B1, HOXB4, HIST1H3C,RASGRF2, TM6SF1, BRCA1, BNC1, ADAMTS1, CDO1, SOX17, TAC1, HOXA7, andMGMT.

Embodiment 268

The method according to any one of embodiments 261-266, wherein saidcancer is pancreatic cancer and said one or more gene promoters comprisethe promoters of one, two, three, or four genes selected from the groupconsisting of ADAMTS1, and BNC1.

Embodiment 269

The method of embodiment 268, wherein said one or more gene promoterscomprise the promoter of ADAMTS1.

Embodiment 270

The method according to any one of embodiments 268-269, wherein said oneor more gene promoters comprise the promoter of BNC1.

Embodiment 271

The method according to any one of embodiments 261-266, wherein saidcancer is breast cancer and said one or more gene promoters comprise thepromoters of one, two, three, four, five, or all genes selected from thegroup consisting of BRCA1, RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2,and TM6SF1.

Embodiment 272

The method of embodiment 271, wherein said one or more gene promoterscomprise the promoter of BRCA1.

Embodiment 273

The method according to any one of embodiments 271-272, wherein said oneor more gene promoters comprise the promoter of RASSF1A.

Embodiment 274

The method according to any one of embodiments 271-273, wherein said oneor more gene promoters comprise the promoter of AKRIB1.

Embodiment 275

The method according to any one of embodiments 271-274, wherein said oneor more gene promoters comprise the promoter of HOXB4.

Embodiment 276

The method according to any one of embodiments 271-275, wherein said oneor more gene promoters comprise the promoter of HIST1H3C.

Embodiment 277

The method according to any one of embodiments 271-276, wherein said oneor more gene promoters comprise the promoter of RASGRF2.

Embodiment 278

The method according to any one of embodiments 271-277, wherein said oneor more gene promoters comprise the promoter of TM6SF1.

Embodiment 279

The method according to any one of embodiments 261-266, wherein saidcancer is breast cancer and said one or more gene promoters comprise thepromoter of BRCA1.

Embodiment 280

The method according to any one of embodiments 261-266, wherein saidcancer is lung cancer and said one or more gene promoters comprise thepromoters of one, two, three, for all genes selected from the groupconsisting of CDO1, SOX17, TAC1, and HOXA7.

Embodiment 281

The method of embodiment 280, wherein said one or more gene promoterscomprise the promoter of CDO1.

Embodiment 282

The method according to any one of embodiments 280-281, wherein said oneor more gene promoters comprise the promoter of SOX17.

Embodiment 283

The method according to any one of embodiments 280-282, wherein said oneor more gene promoters comprise the promoter of TAC1.

Embodiment 284

The method according to any one of embodiments 280-283, wherein said oneor more gene promoters comprise the promoter of HOXA7.

Embodiment 285

The method according to any one of embodiments 261-266, wherein saidcancer is brain cancer and said one or more gene promoters comprise thepromoter of MGMT.

Embodiment 286

A method of converting cytosine residues in a DNA to uracil, whileleaving 5-methylcytosine residues substantially unaffected, said methodcomprising:

contacting a sample comprising DNA with DABSO to convert said DNA;

desulphonating the converted DNA, to produce a DNA in which cytosineresidues are converted to uracil, but 5-methylcytosine residuessubstantially unaffected.

Embodiment 287

The method of embodiment 286, wherein said contacting comprisescontacting said DNA with DABSO at a concentration ranging from about 2 Mup to about 5 M.

Embodiment 288

The method of embodiment 286, wherein said contacting comprisescontacting said DNA with DABSO at a concentration of about 2.5 M.

Embodiment 289

The method according to any one of embodiments 286-288, wherein saidDABSO is dissolved in an alkaline aqueous solution.

Embodiment 290

The method of embodiment 289, wherein said DABSO is dissolved in asolution comprising KOH.

Embodiment 291

The method according to any one of embodiments 286-290, wherein saidcontacting comprises heating the DABSO/DNA solution to a temperatureranging from about 55° C. to about 90° C.

Embodiment 292

The method according to any one of embodiments 286-291, wherein saidDABSO is reacted with the DNA for a period of time ranging from about 15minutes up to about 90 minutes.

Embodiment 293

The method according to any one of embodiments 286-292, wherein saiddesulphonating comprises contacting said converted DNA with an alkalinereagent.

Embodiment 294

The method of embodiment 293, wherein said alkaline reagent comprisesKOH.

Embodiment 295

The method according to any one of embodiments 286-294, wherein saidconversion and/or desulphonation is performed on the DNA bound to acolumn.

Embodiment 296

The method according to any one of embodiments 286-294, wherein saidconversion and/or desulphonation is performed on the DNA in solution.

Embodiment 297

A method of analyzing DNA methylation, said method comprising:

providing a DNA sample;

converting DNA in said sample according to the method of any one ofembodiments 286-296; and

performing methylation specific PCR and/or nucleic acid sequencing,and/or high resolution melting analysis (HRM) on the converted nucleicacid to determine the methylation of said nucleic acid.

Embodiment 298

The method of embodiment 297, wherein said providing a DNA samplecomprises preparing a sample according to any one of embodiments 228-234or according to any one of embodiments 243-252.

Embodiment 299

A kit for detection of methylation state of a DNA, said kit comprising:

a container containing a conversion reagent comprising DABSO; and

a container containing a desulphonation reagent.

Embodiment 300

The kit of embodiment 299, wherein said kit comprises a columncomprising an affinity matrix.

Embodiment 301

The kit according to any one of embodiments 299-300, wherein said kitcomprises a container containing a binding buffer.

Embodiment 302

The kit according to any one of embodiments 299-301, wherein said kitcomprises a container containing an elution buffer.

Embodiment 303

The kit according to any one of embodiments 299-302, wherein said kitcomprises a container containing a wash buffer.

Embodiment 304

The kit according to any one of embodiments 299-303, wherein said kitcomprises a container containing a lysis solution according to any oneof embodiments 169-171, and/or a container containing a lysis solutionaccording to any one of embodiments 172-174.

Embodiment 305

The kit according to any one of embodiments 299-304, wherein said kitcomprises a cartridge according to any one of embodiments 143-155 and/ora cartridge according to any one of embodiments 155-166.

Embodiment 306

The kit according to any one of embodiments 299-305, said kit comprisinginstructional materials teaching the use of said kit to convert a nucleiacid for determination of the methylation state of said nucleic acid.

Embodiment 307

A set of primers and probes for the determination of methylation of MGMTusing a nested PCR reaction, said set comprising the following primersand probes:

an external forward primer comprising the nucleotide sequenceGTTTT(T*)AGAAYG(T*)TTTGYGTTT (SEQ ID NO:263);

an external reverse primer comprising the nucleotide sequenceAAAAAAC(T*)CCRCACTCTTCC (SEQ ID NO:265);

an internal forward primer comprising the nucleotide sequenceTTTCGACGTTCGTAGGTTTTCGC (SEQ ID NO:266);

an internal reverse primer comprising the nucleotide sequenceGCACTCTTCCGAAAACGAAACG (SEQ ID NO:267); and

a probe comprising the nucleotide sequencefluor-CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker (SEQ ID NO: 268).

Embodiment 308

A set of primers and probes for the determination of methylation of ACTB(e.g., as a control) using a nested PCR reaction, said set comprisingthe following primers and probes:

an external forward primer (102) comprising the nucleotide sequenceGTGATGGAGGAGGTTTAGTAAGTT (SEQ ID NO:103);

an external reverse primer (103) comprising the nucleotide sequenceCCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO:104);

an internal forward primer (148) comprising the nucleotide sequenceGGTTTAGTAAGTTTTTTGGATTGTG (SEQ ID NO:149);

an internal reverse primer (149) comprising the nucleotide sequenceCCTTAAAAATTACAAAAACCACAAC (SEQ ID NO:150); and

a probe (178) comprising the nucleotide sequencefluor-CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker (SEQ ID NO:179).

Embodiment 309

A set of primers and probes for the determination of methylation of MGMTusing a nested PCR reaction with determination of the methylation ofACTB as a control, comprising the primers and probes of embodiment 307and the primers and probes of embodiment 308.

Embodiment 310

A method of determining the methylation of MGMT using methylationspecific PCR said method comprising:

providing a converted (e.g., bisulfite converted) DNA containing apromoter region of the MGMT gene;

performing methylation specific PCR for MGMT methylation using a nestedPCR reaction comprising the following primers and probes:

-   -   an external forward primer comprising the nucleotide sequence        GTT TT(T*)AGAAYG(T*)TTTGYGTTT(SEQ ID NO:263);    -   an external reverse primer comprising the nucleotide sequence        AAAAAAC(T*)CCRCACTCTTCC (SEQ ID NO:265);    -   an internal forward primer comprising the nucleotide sequence        TTTCGACGTTCGTAGGTTTTCGC (SEQ ID NO:266);    -   an internal reverse primer comprising the nucleotide sequence        GCACTCTTCCGAAAACGAAACG (SEQ ID NO:267); and    -   a probe comprising the nucleotide sequence        fluor-CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker (SEQ ID NO: 268);        and

detecting and/or quantifying the PCR amplification product to providedetermine methylation of said MGMT gene.

Embodiment 311

The method of embodiment 310, wherein said method further comprises:

providing a converted (e.g., bisulfite converted) DNA containing apromoter region of the ACTB gene (e.g., as a control);

performing methylation specific PCR for ACTB methylation using a nestedPCR reaction comprising the following primers and probes:

-   -   an external forward primer comprising the nucleotide sequence        GTGATGGAGGAGGTTTAGTAAGTT (SEQ ID NO:103);    -   an external reverse primer comprising the nucleotide sequence        CCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO:104);    -   an internal forward primer comprising the nucleotide sequence        GGTTTAGTAAGTTTTTTGGATTGTG (SEQ ID NO:149);    -   an internal reverse primer comprising the nucleotide sequence        CCTTAAAAATTACAAAAACCACAAC (SEQ ID NO:150); and    -   a probe comprising the nucleotide sequence        fluor-CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker (SEQ ID        NO:179); and

detecting and/or quantifying the PCR amplification product to providedetermine methylation of said ACTB gene.

Embodiment 312

The method according to any one of embodiments 310-311, wherein saidmethylation specific PCR for MGMT methylation and said methylationspecific PCR for ACTB methylation are performed in a single multiplexPCR reaction.

Embodiment 313

The method according to any one of embodiments 310-312, wherein saidmethylation specific PCR is performed using a cartridge according to anyone of embodiments 92-132.

Embodiment 314

The method of embodiment 313, wherein: said providing a converted DNAcontaining a promoter region of the MGMT gene comprises introducing anunconverted DNA containing a promoter region of the MGMT gene into saidcartridge and operating said cartridge to convert said DNA in saidcartridge using a conversion reagent; and/or said providing a convertedDNA containing a promoter region of the ACTB gene comprises introducingan unconverted DNA containing a promoter region of the ACTB gene intosaid cartridge and operating said cartridge to convert said DNA in saidcartridge using a conversion reagent.

Embodiment 315

The method of embodiment 314, wherein said conversion reagent comprisesa compound selected from the group consisting of ammonium bisulfite,sodium metabisulfite, potassium bisulfite, cesium bisulfite, and DABSO.

Embodiment 316

The method according to any one of embodiments 313-315, wherein saidoperating said cartridge comprises heating said DNA and said conversionreagent in a thermocycling channel or chamber that is later used toperform said nested PCR reaction.

Embodiment 317

A set of cartridges for determining the methylation state of a nucleicacid, said set of cartridges comprising: a first cartridge comprising:

a sample receiving chamber;

a column comprising a first matrix material;

a temperature controlled channel or chamber;

a sample removal chamber; and

a plurality of chambers containing reagents and/or buffers, wherein whenin use at least one of said chambers contains a bisulfite reagent; and

a second cartridge comprising:

a sample receiving chamber;

a column comprising a second matrix material;

a temperature controlled channel or chamber; and

a plurality of chambers containing reagents and/or buffers, wherein whenin use at least one of said chambers contains a desulphonation and/orelution reagent.

Embodiment 318

The set of cartridges of embodiment 317, wherein the temperaturecontrolled channel or chamber in said first cartridge is a thermocyclingchannel or chamber.

Embodiment 319

The set of cartridges according to any one of embodiments 317-318,wherein the temperature controlled channel or chamber in said secondcartridge is a thermocycling channel or chamber.

Embodiment 320

The set of cartridges according to any one of embodiments 317-319,wherein said bisulfite reagent comprises a compound selected from thegroup consisting of ammonium bisulfite, sodium metabisulfite, potassiumbisulfite, cesium bisulfite, and DABSO.

Embodiment 321

The set of cartridges of embodiment 320, wherein said bisulfite reagentcomprises ammonium bisulfite.

Embodiment 322

The set of cartridges according to any one of embodiments 317-321,wherein said bisulfite is provided in a reagent mix comprisingscavengers to prevent sulfite oxidation and/or catalysts.

Embodiment 323

The set of cartridges of embodiment 322, wherein said bisulfite isprovided in a reagent mix comprising scavengers selected from the groupconsisting of Trolox and hydroquinone.

Embodiment 324

The set of cartridges according to any one of embodiments 322-323,wherein said bisulfite is provided in a reagent mix comprisingpolyamines as catalysts.

Embodiment 325

The set of cartridges according to any one of embodiments 317-324,wherein said first cartridge is configured for the bisulfite reagent tobe added to the cartridge at or near the time the sample is placed inthe cartridge.

Embodiment 326

The set of cartridges according to any one of embodiments 317-325,wherein the bisulfite reagent is provided as a component in one of saidplurality of chambers in said first the cartridge.

Embodiment 327

The set of cartridges according to any one of 317-326, wherein saidfirst matrix material and/or said second matrix material, independentlycomprise a material is selected from the group consisting of glass orsilica, an ion exchange resin, and hydroxyapatite.

Embodiment 328

The set of cartridges of embodiment 327, wherein said first matrixmaterial comprises glass fibers.

Embodiment 329

The set of cartridges according to any one of 327-328, wherein saidsecond matrix material comprises glass fibers.

Embodiment 330

The set of cartridges according to any one of embodiments 317-329,wherein said first cartridge comprises two sample receiving chambers.

Embodiment 331

The set of cartridges according to any one of 317-330, wherein at leastone chamber comprising the plurality of chambers in said secondcartridge contains PCR primers, and/or PCR probes, and/or a PCR mastermix.

Embodiment 332

The set of cartridges according to any one of 317-331, wherein, whensaid cartridge is in use, a chamber comprising the plurality of chambersin said first cartridge contains GTC-EtOH in a buffer.

Embodiment 333

The set of cartridges according to any one of 317-332, wherein, whensaid cartridge is in use, a chamber comprising the plurality of chambersin said second cartridge contains GTC-EtOH in a buffer.

Embodiment 334

The set of cartridges according to any one of 332-333, wherein the firstcartridge is configured for addition of GTC-ETOH in a buffer at or nearthe time the sample is placed into the cartridge.

Embodiment 335

The set of cartridges according to any one of 332-333, wherein theGTC-ETOH in a buffer is provided as a component in a chamber comprisingthe plurality of chambers of the first cartridge.

Embodiment 336

The set of cartridges according to any one of 332-335, wherein thesecond cartridge is configured for addition of GTC-ETOH in a buffer ator near the time the sample is placed into the cartridge.

Embodiment 337

The set of cartridges according to any one of 332-336, wherein theGTC-ETOH in a buffer is provided as a component in a chamber comprisingthe plurality of chambers of the second cartridge.

Embodiment 338

The set of cartridges according to any one of embodiments 317-337,wherein said second cartridge comprises one or more chambers containingone or more reagents selected from the group consisting of methylationspecific PCR primers, methylation specific PCR probes, PCR enzyme(s),and PCR reaction buffer.

Embodiment 339

The set of cartridges of embodiment 338, wherein said second cartridgecontains at least two chambers containing one or more reagents selectedfrom the group consisting of methylation specific PCR primers,methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer.

Embodiment 340

The set of cartridges according to any one of embodiments 317-339,wherein said second cartridge contains at least one chamber containingprimers and probes for detection of methylation of a forward strand of aconverted DNA.

Embodiment 341

The set of cartridges according to any one of embodiments 317-340,wherein said second cartridge contains at least one chamber containingprimers and probes for detection of methylation of a reverse strand of aconverted DNA.

Embodiment 342

The set of cartridges according to any of embodiments 338-341, whereinsaid PCR primers, and/or probes, and/or enzymes are provided as beads.

Embodiment 343

The set of cartridges according to any one of embodiments 317-342,wherein:

in said first cartridge said sample receiving chamber, said column, saidplurality of chambers, said sample removal chamber, and saidtemperature-controlled heating channel or chamber, are selectively influid communication; and/or

in said second cartridge said sample receiving chamber, said column,said plurality of chambers, and said temperature-controlled heatingchannel or chamber, are selectively in fluid communication.

Embodiment 344

The set of cartridges of embodiment 343, wherein

in said first cartridge, said sample receiving chamber, said column,said plurality of chambers, said sample removal chamber, and saidtemperature controlled channel or chamber, are selectively in fluidcommunication by microfluidic channels and valves; and/or

in said second cartridge, said sample receiving chamber, said column,said plurality of chambers, and said temperature controlled channel orchamber, are selectively in fluid communication by microfluidic channelsand valves.

Embodiment 345

The set of cartridges of embodiment 343, wherein

in said first cartridge, said sample receiving chamber, said column,said plurality of chambers, said sample removal chamber, and saidtemperature controlled channel or chamber or a port into saidtemperature controlled channel or chamber, are disposed around a centralvalve and selectively in fluid communication with a channel in saidcentral valve, wherein said central valve is configured to accommodate aplunger that is capable of drawing fluid into or out of a chamber influid communication with said central valve; and/or

in said second cartridge, said sample receiving chamber, said column,said plurality of chambers, and said temperature controlled channel orchamber or a port into said temperature controlled channel or chamber,are disposed around a central valve and selectively in fluidcommunication with a channel in said central valve, wherein said centralvalve is configured to accommodate a plunger that is capable of drawingfluid into or out of a chamber in fluid communication with said centralvalve.

Embodiment 346

The set of cartridges according to any one of embodiments 317-345,wherein said first cartridge is configured so that, when in use, saidfirst cartridge comprises:

a first chamber containing a sample;

a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH)solution;

a third chamber containing a bisulfite reagent;

a fourth chamber containing a Tris buffer;

a fifth chamber polyethyleneglycol (PEG); and

a sixth chamber containing KOH.

Embodiment 347

The set of cartridges of embodiment 346, wherein said first chamber insaid first cartridge contains said sample in a GTC-EtOH-Tweenextraction/precipitation reagent.

Embodiment 348

The set of cartridges according to any one of embodiments 346-347,wherein said a second chamber containing a guanidiniumthiosulfate-ethanol (GTC-EtOH) solution contains GTC-Tris (pH 7),ethanol.

Embodiment 349

The set of cartridges according to any one of embodiments 317-336,wherein said second cartridge is configured so that, when in use, saidsecond cartridge comprises:

a first chamber containing a sample;

a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH)solution;

a third chamber containing a Tris buffer;

a fourth chamber containing polyethylene glycol (PEG);

a fifth chamber containing KOH; and

a sixth chamber containing a PCR enzyme bead.

Embodiment 350

The set of cartridges of embodiment 349, wherein said second cartridgecomprises a seventh chamber containing a Tris bead, and an enzyme bead.

Embodiment 351

The set of cartridges according to any one of embodiments 349-350,wherein said a second chamber containing a guanidiniumthiosulfate-ethanol (GTC-EtOH) solution contains GTC-Tris (pH 7),ethanol.

Embodiment 352

The set of cartridges according to any one of embodiments 317-351,wherein said second cartridge comprises a chamber containing PCR primersand/or probes and/or PCR enzymes.

Embodiment 353

The set of cartridges according to any one of embodiments 317-352,wherein said second cartridge comprises an eighth chamber alsocontaining PCR primers and/or probes and/or PCR enzymes.

Embodiment 354

The set of cartridges according to any one of embodiments 317-353,wherein said second cartridge comprises one or more chambers containingprimers specific for bisulfite-converted methylated and/or unmethylatedsequences.

Embodiment 355

The set of cartridges according to any one of embodiments 317-354,wherein said second cartridge comprises one or more chambers containingreagents for TaqMan PCR reactions.

Embodiment 356

The set of cartridges according to any one of embodiments 317-355,wherein said second cartridge comprises one or more chambers containingone or more fluorescent probes that are markers for amplified methylatedsequences and/or one or more fluorescent probes that are markers foramplified unmethylated sequences.

Embodiment 357

The set of cartridges of embodiment 356, wherein said probes comprise afluorescent reporter dye and a quencher dye, where the probes provides asignal upon cleavage by the 5′ to 3′ nuclease activity of Taq DNApolymerase.

Embodiment 358

The set of cartridges according to any one of embodiments 356-357,wherein said second cartridge comprises a plurality of probes eachspecific to a different methylated region in an amplified region ofinterest.

Embodiment 359

The set of cartridges according to any one of embodiments 356-357,wherein said second cartridge comprises a single probe specific to amethylated region in an amplified region of interest.

Embodiment 360

The set of cartridges according to any one of embodiments 356-357,wherein said second cartridge comprises a plurality of probes eachspecific to the same methylated region in an amplified region ofinterest.

Embodiment 361

The set of cartridges according to any one of embodiments 317-360,wherein said second cartridge contains primers and/or probes todetermine methylation of a promoter region of a gene selected from thegroup consisting of MGMT, RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4,RASGRF2, TM6SF1, and AKR1B1.

Embodiment 362

The set of cartridges according to any one of embodiments 317-360,wherein said second cartridge contains one or more primers shown inTables 5, 9, or 10, and/or one or more probes shown in Tables 5, 9, or10.

Embodiment 363

The set of cartridges of embodiment 362, wherein said second cartridgecontains the following probes and primers for determining methylation ofMGMT using a nested PCR reaction:

an external forward primer (248b) comprising the nucleotide sequenceGTTTT(T*)AGAAYG(T*)TTTGYGTTT (SEQ ID NO:263);

an external reverse primer (249b) comprising the nucleotide sequence:AAAAAAC(T*)CCRCACTCTTCC (SEQ ID NO:265);

an internal forward primer (250) comprising the nucleotide sequenceTTTCGACGTTCGTAGGTTTTCGC (SEQ ID NO:266);

an internal reverse primer (251) comprising the nucleotide sequenceGCACTCTTCCGAAAACGAAACG (SEQ ID NO:267); and

a probe (252a) comprising the nucleotide sequencefluor-CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker (SEQ ID NO:268).

Embodiment 364

The set of cartridges according to any one of embodiments 362-363,wherein said second cartridge contains the following probes and primersfor determining methylation of ACTB (e.g., as a control) using a nestedPCR reaction:

an external forward primer (102) comprising the nucleotide sequenceGTGATGGAGGAGGTTTAGTAAGTT (SEQ ID NO:103);

an external reverse primer (103) comprising the nucleotide sequenceCCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO:104);

an internal forward primer (148) comprising the nucleotide sequenceGGTTTAGTAAGTTTTTTGGATTGTG (SEQ ID NO:149);

an internal reverse primer (149) comprising the nucleotide sequenceCCTTAAAAATTACAAAAACCACAAC (SEQ ID NO:150); and

a probe (178) comprising the nucleotide sequencefluor-CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker (SEQ ID NO:179).

Embodiment 365

The set of cartridges according to any one of embodiments 317-360,wherein said second cartridge comprises one or more chambers containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters.

Embodiment 366

The set of cartridges of embodiment 365, wherein said second cartridgecomprises one or more chambers containing beads that provide PCR primersand probes to detect methylation of one or more gene promoters whosemethylation state is a marker for a cancer selected from the groupconsisting of breast cancer, pancreatic cancer, prostate cancer, braincancer, and lung cancer.

Embodiment 367

The set of cartridges according to any of embodiments 365-366, whereinsaid chamber containing beads that provide PCR primers and probes todetect methylation of one or more gene promoters comprises beads thatprovide PCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for a cancer selected fromthe group consisting of breast cancer, pancreatic cancer, prostatecancer, brain cancer, and lung cancer.

Embodiment 368

The set of cartridges of embodiment 367, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes for a nested PCR reaction.

Embodiment 369

The set of cartridges of embodiment 368, wherein said nested PCRcomprises a first PCR reaction specific for converted DNA and a secondPCR reaction specific for methylated CpGs.

Embodiment 370

The set of cartridges according to any one of embodiments 349-369,wherein said chamber containing beads that provide PCR primers andprobes chamber contains beads that provide PCR primers and probes todetect methylation of a forward strand of converted DNA.

Embodiment 371

The set of cartridges according to any one of embodiments 349-370,wherein said chamber containing beads that provide PCR primers andprobes chamber contains beads that provide PCR primers and probes todetect methylation of a reverse strand of converted DNA.

Embodiment 372

The set of cartridges according to any of embodiments 349-371, whereinsaid chamber containing beads that provide PCR primers and probes todetect methylation of one or more gene promoters comprises beads thatprovide PCR primers and probes to detect methylation of the promoters ofone or more genes selected from the group consisting of RASSF1A, AKR1B1,HOXB4, HIST1H3C, RASGRF2, TM6SF1, BRCA1, BNC1, ADAMTS1, CDO1, SOX17,TAC1, HOXA7, and MGMT.

Embodiment 373

The set of cartridges according to any of embodiments 349-372, whereinsaid chamber containing beads that provide PCR primers and probes todetect methylation of one or more gene promoters comprises beads thatprovide PCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for pancreatic cancer.

Embodiment 374

The set of cartridges of embodiment 373, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes to detect methylation of the promoters of ADAMTS1, and/or BNC1.

Embodiment 375

The set of cartridges of embodiment 374, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes to detect methylation of the promoter of ADAMTS1.

Embodiment 376

The set of cartridges according to any one of embodiments 374-375,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of BNC1.

Embodiment 377

The set of cartridges of embodiment 374, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide one or more PCRprimers and/or probes for ADAMTS1 and/or BNC1 shown in Tables 5, or 10.

Embodiment 378

The set of cartridges according to any of embodiments 349-372, whereinsaid chamber containing beads that provide PCR primers and probes todetect methylation of one or more gene promoters comprises beads thatprovide PCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for breast cancer.

Embodiment 379

The set of cartridges of embodiment 378, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes to detect methylation of the promoters of one, two, three, four,five, or all genes selected from the group consisting of BRCA1, RASSF1A,AKR1B1, HOXB4, HIST1H3C, RASGRF2, and TM6SF1.

Embodiment 380

The set of cartridges of embodiment 379, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes to detect methylation of the promoter of BRCA1.

Embodiment 381

The set of cartridges according to any one of embodiments 379-380,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of RASSF1A.

Embodiment 382

The set of cartridges according to any one of embodiments 379-381,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of AKRIB1.

Embodiment 383

The set of cartridges according to any one of embodiments 379-382,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of HOXB4.

Embodiment 384

The set of cartridges according to any one of embodiments 379-383,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of HIST1H3C.

Embodiment 385

The set of cartridges according to any one of embodiments 379-384,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of RASGRF2.

Embodiment 386

The set of cartridges according to any one of embodiments 379-385,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of TM6SF1.

Embodiment 387

The set of cartridges according to any one of embodiments 379-386,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide one or more PCR primers and/or one or more PCR probesshown in Tables 5, or 9.

Embodiment 388

The set of cartridges of embodiment 378, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes to detect methylation of the promoters of BRCA1.

Embodiment 389

The set of cartridges according to any of embodiments 349-372, whereinsaid chamber containing beads that provide PCR primers and probes todetect methylation of one or more gene promoters comprises beads thatprovide PCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for lung cancer.

Embodiment 390

The set of cartridges of embodiment 389, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes to detect methylation of the promoters of one, two, three, or allgenes selected from the group consisting of CDO1, SOX17, TAC1, andHOXA7.

Embodiment 391

The set of cartridges of embodiment 390, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes to detect methylation of the promoter of CDO1.

Embodiment 392

The set of cartridges according to any one of embodiments 390-391,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of SOX17.

Embodiment 393

The set of cartridges according to any one of embodiments 390-392,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of TAC1.

Embodiment 394

The set of cartridges according to any one of embodiments 390-393,wherein said chamber containing beads that provide PCR primers andprobes to detect methylation of one or more gene promoters comprisesbeads that provide PCR primers and probes to detect methylation of thepromoter of HOXA7.

Embodiment 395

The set of cartridges according to any of embodiments 349-372, whereinsaid chamber containing beads that provide PCR primers and probes todetect methylation of one or more gene promoters comprises beads thatprovide PCR primers and probes to detect methylation of one or more genepromoters whose methylation state is a marker for brain cancer.

Embodiment 396

The set of cartridges of embodiment 395, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide PCR primers andprobes to detect methylation of the promoter of MGMT.

Embodiment 397

The set of cartridges of embodiment 396, wherein said chamber containingbeads that provide PCR primers and probes to detect methylation of oneor more gene promoters comprises beads that provide one or more PCRprimers and/or probes for MGMT shown in Tables 5, or 10.

Embodiment 398

The set of cartridges of embodiment 397, wherein said cartridge containsthe following probes and primers for determining methylation of MGMTusing a nested PCR reaction:

an external forward primer (248b) comprising the nucleotide sequenceGTTTT(T*)AGAAYG(T*)TTTGYGTTT (SEQ ID NO:263);

an external reverse primer (249b) comprising the nucleotide sequenceAAAAAAC(T*)CCRCACTCTTCC (SEQ ID NO:265);

an internal forward primer (250) comprising the nucleotide sequenceTTTCGACGTTCGTAGGTTTTCGC (SEQ ID NO:266);

an internal reverse primer (251) comprising the nucleotide sequenceGCACTCTTCCGAAAACGAAACG (SEQ ID NO:267); and

a probe (252a) comprising the nucleotide sequencefluor-CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker (SEQ ID NO: 268).

Embodiment 399

The set of cartridges according to any one of embodiments 397-398,wherein said cartridge contains the following probes and primers fordetermining methylation of ACTB (e.g., as a control) using a nested PCRreaction:

an external forward primer (102) comprising the nucleotide sequence:

GTGATGGAGGAGGTTTAGTAAGTT (SEQ ID NO:103);

an external reverse primer (103) comprising the nucleotide sequence:

CCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO:104);

an internal forward primer (148) comprising the nucleotide sequence:

GGTTTAGTAAGTTTTTTGGATTGTG (SEQ ID NO:149);

an internal reverse primer (149) comprising the nucleotide sequence:

CCTTAAAAATTACAAAAACCACAAC (SEQ ID NO:150); and

an probe (178) comprising the nucleotide sequence:

fluor-CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker (SEQ ID NO:179).

Embodiment 400

A system for determining the methylation of a nucleic acid in abiological sample, said system comprising:

an enclosure configured to contain one or more sample processingmodules, each sample processing module configured to hold a removablecartridge first cartridge and/or second cartridge of said set ofcartridges according to any one of embodiments 317-399;

where said system is configured to:

-   -   operate the sample processing modules to perform sample        processing to operate the first cartridge of said set of        cartridges to perform a bisulfite conversion of a nucleic acid        in a sample introduced into said first cartridge; and/or

to perform a desulphonation and to determine methylation of one or moretarget nucleic acids within a corresponding removable sample cartridge.

Embodiment 401

The system of embodiment 400, wherein said system is configured tocontain one sample processing module.

Embodiment 402

The system of embodiment 400, wherein said system is configured tocontain at least two sample processing modules, or at least 4 sampleprocessing modules, or at least 8 sample processing modules, or at least12 sample processing modules, or at least 16 sample processing modules,or at least 20 sample processing modules, or at least 24 sampleprocessing modules, or at least 28 sample processing modules, or atleast 32 sample processing modules, or at least 64 sample processingmodules, or at least 128 sample processing modules.

Embodiment 403

The system according to any one of embodiments 400-402, wherein saidmodules comprise one or more heating plates to heat a temperaturecontrolled chamber or channel in said cartridge.

Embodiment 404

The system according to any one of embodiments 400-403, wherein saidmodules comprise a fan configured to cool a temperature controlledchannel or chamber in said cartridge.

Embodiment 405

The system according to any one of embodiments 400-404, wherein saidmodules comprise circuitry to pass information (e.g., opticalinformation) to a computer for analysis.

Embodiment 406

The system according to any one of embodiments 400-405, wherein saidmodules comprise optical blocks to provide excitation and/or detectionof one or more optical signals produced by reactions in said cartridge.

Embodiment 407

The system according to any one of embodiments 400-406, wherein saidsystem is configured to operate said first cartridge of said set ofcartridges to:

bind a sample to a column;

elute DNA from the column and combine said DNA with a conversionreagent;

heat the DNA/conversion reagent solution in a reaction chamber or tubeto produce converted DNA; and

to deliver the converted DNA to a sample removal chamber in the firstcartridge.

Embodiment 408

The system according to any one of embodiments 400-407, wherein saidsystem is configured to operate said second cartridge in said set ofcartridges to:

bind the converted DNA to a column;

wash, rinse, and elute the converted DNA;

elute the DNA from the column; and

defulphonate the converted DNA.

Embodiment 409

The system of embodiment 408, wherein said system is configured tooperate said second cartridge in said set of cartridges to perform PCRon the eluted desulphonated DNA in a reaction chamber or tube.

Embodiment 410

A method of determining the methylation state of a nucleic acid, saidmethod comprising: providing a biological sample in a sample chamber ofa first cartridge in a set of cartridges according to any one ofembodiments 317-399; and operating said first cartridge to:

bind DNA in said sample to said first matrix material;

wash the bound DNA; elute the bound DNA off of the matrix material;

combine the eluted DNA with said bisulfite reagent;

heat the mixture of DNA and bisulfite reagent in said temperaturecontrolled channel or chamber perform a bisulfite conversion of saidDNA; and

deliver the bisulfite-converted DNA into the sample removal chamber ofsaid first cartridge.

Embodiment 411

The method of embodiment 410, wherein said method further comprise:

providing bisulfite converted DNA in a sample chamber of a secondcartridge in a set of cartridges according to any one of embodiments317-399; and

operating said second cartridge to:

-   -   bind said bisulfite converted DNA to said second matrix        material;    -   wash the bound bisulfite-converted DNA;    -   elute the washed bisulfite-converted DNA from said second matrix        material; and    -   desulphonate the bisulfite converted DNA.

Embodiment 412

The method of embodiment 411, wherein said second cartridge is operatedto elute the bisulfite-converted DNA from said second matrix materialbefore desulphonation.

Embodiment 413

The method of embodiment 411, wherein said second cartridge is operatedto elute the bisulfite-converted DNA from said second matrix materialafter or during desulphonation.

Embodiment 414

The method according to any one of embodiments 411-413, wherein saidmethod comprises operating said second cartridge to perform methylationspecific PCR and/or nucleic acid sequencing, and/or high resolutionmelting analysis (HRM) on said converted nucleic acid to determine themethylation of said nucleic acid.

Embodiment 415

The method according to any one of embodiments 410-414, wherein saidsample comprises one or more samples selected from the group consistingof a cell, a tissue, and a biological fluid containing a nucleic acid.

Embodiment 416

The method of embodiment 415, wherein said biological sample comprises abiological fluid selected from the group consisting of whole blood,plasma, serum, saliva, mucus, urine, sputum, pancreatic juice, andcerebrospinal fluid.

Embodiment 417

The method of embodiment 415, wherein said biological sample comprises asample selected from the group consisting of a tissue sample, a formalinfixed paraffin embedded (FFPE) tissue, fresh frozen tissue, fine needleaspirates (FNA), and a core biopsy.

Embodiment 418

The method according to any one of embodiments 410-417, wherein saidmethod comprises contacting said biological sample with a lysissolution.

Embodiment 419

The method according to any one of embodiments 410-418, whereinoperating said first cartridge comprises introducing said firstcartridge into a sample processing module in a system according to anyone of embodiments 400-409.

Embodiment 420

The method according to any one of embodiments 410-419, whereinoperating said second cartridge comprises introducing said secondcartridge into a sample processing module in a system according to anyone of embodiments 400-409.

Embodiment 421

The method according to any one of embodiments 410-420, wherein saidproviding a biological sample in a sample chamber of a first cartridgecomprises loading a sample into one or more sample receiving chambers insaid first cartridge.

Embodiment 422

The method according to any one of embodiments 410-421, wherein saidproviding bisulfite converted DNA in a sample chamber of a secondcartridge comprises transferring bisulfite-converted DNA from a sampleremoval chamber in said first cartridge into a sample receiving chamberof said second cartridge.

Embodiment 423

The method according to any one of embodiments 410-422, wherein saideluting the bound DNA in said first cartridge comprises eluting anddenaturing said DNA using a low concentration of potassium hydroxide orother base.

Embodiment 424

The method of embodiment 423, wherein said eluting the bound DNA in saidfirst cartridge comprises eluting and denaturing said DNA with analkaline solution with a pH greater than about pH 10.5.

Embodiment 425

The method of embodiment 423, wherein said eluting the bound DNA in saidfirst cartridge comprises eluting and denaturing said DNA with analkaline solution with a pH greater than about pH 12.

Embodiment 426

The method of embodiments 424-425, wherein said alkaline solution is a10-15 mM KOH solution.

Embodiment 427

The method according to any one of embodiments 410-426, wherein saidcombining the eluted DNA with a bisulfite reagent in said firstcartridge comprises incubating the DNA in an ammonium bisulfite solutionhaving a concentration that ranges from about 6M to about 7M.

Embodiment 428

The method of embodiment 427, wherein said combining the eluted DNA witha bisulfite reagent in said first cartridge comprises incubating the DNAin an ammonium bisulfite solution having a concentration of about 6.5M.

Embodiment 429

The method of embodiment 428, wherein said combining the eluted DNA witha bisulfite reagent in said first cartridge comprises transferring theDNA in a concentrated bisulfite solution into a temperature controlledchannel or chamber in said first cartridge and heating said mixture.

Embodiment 430

The method of embodiment 429, wherein said incubating comprisesthermally cycling the concentrated bisulfite solution from a temperatureof about 60° C. to about 95° C.

Embodiment 431

The method according to any one of embodiments 411-430, wherein saidbinding the bisulfite converted DNA to the second matrix materialcomprises mixing the DNA-bisulfite solution with fresh GTC-EtOH in saidsecond column, and dispensing the solution over said second matrixmaterial.

Embodiment 432

The method of embodiment 431, wherein said method comprises washing theDNA in said second matrix material with fresh GTC-EtOH, and then a rinsesolution.

Embodiment 433

The method of embodiment 432, wherein said rinse solution comprisesPEG200.

Embodiment 434

The method according to any one of embodiments 411-433, wherein saiddesulphonating the converted DNA comprises eluting the DNA from saidsecond matrix material with a high pH desulphonation buffer andincubating said solution.

Embodiment 435

The method of embodiment 434, wherein said incubating is for a period oftime ranging from about 1 minute to about 1 hour, or from about 5minutes to about 30 minutes, or from about 10 minutes to about 20minutes, or for about 15 minutes.

Embodiment 436

The method of embodiments 434-435, wherein said high pHdesulphonation/elution buffer comprises KOH.

Embodiment 437

The method according to any one of embodiments 411-436, wherein saidmethod comprises operating said second cartridge to perform one or morePCR reactions using said converted DNA as a template.

Embodiment 438

The method of embodiment 437, wherein methylation specific PCR isperformed to determine methylation of target nucleic acid sequences.

Embodiment 439

The method of embodiment 438, wherein said methylation specific PCR(MSP) is performed using primers specific for methylated sequencesand/or primers specific for unmethylated sequences.

Embodiment 440

The method of embodiment 438, wherein said methylation specific PCRcomprises a MethyLight protocol.

Embodiment 441

The method of embodiment 438, wherein TaqMan PCR reactions are performedwith primers specific for bisulfite-converted methylated and/orunmethylated sequences.

Embodiment 442

The method according to any one of embodiments 438-441, wherein saidmethylation specific PCR (MSP) utilizes one or more fluorescent probesthat are markers for amplified methylated sequences and/or one or morefluorescent probes that are markers for amplified unmethylatedsequences.

Embodiment 443

The method of embodiment 442, wherein said fluorescent probes comprise afluorescent reporter dye and a quencher dye where the probe provides asignal upon cleavage by 5′ to 3′ nuclease activity of Taq DNApolymerase.

Embodiment 444

The method according to any one of embodiments 442-443, wherein amethylation signal is determined by the combined signal for a pluralityof probes each specific to a different methylated region in an amplifiedregion of interest.

Embodiment 445

The method according to any one of embodiments 442-443, wherein amethylation signal is determined by a plurality of probes specific forthe same methylated region in an amplified region of interest.

Embodiment 446

The method according to any one of embodiments 442-443, wherein saidplurality of probes comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more probes.

Embodiment 447

The method according to any one of embodiments 442-443, wherein amethylation signal is determined by a single probe in the amplifiedregion of interest.

Embodiment 448

The method according to any one of embodiments 442-447, wherein saidprobes are run in simplex or multiplex.

Embodiment 449

The method according to any one of embodiments 442-447, wherein saidprobes are run in a multiplex format.

Embodiment 450

The method according to any one of embodiments 442-449, wherein saidprobes are run as a nested PCR reaction.

Embodiment 451

The method according to any one of embodiments 442-450, wherein said PCRreaction comprises a bisulfite contamination control probe that thatundergoes bisulfite-mediated cleavage during PCR if bisulfite is presentin the reaction.

Embodiment 452

The method according to any one of embodiments 411-451, wherein PCR isperformed for one or more mutated genes.

Embodiment 453

The method according to any one of embodiments 411-452, wherein PCR isperformed for unconverted DNA as a control.

Embodiment 454

The method according to any one of embodiments 411-453, wherein PCR isperformed for converted DNA as a control.

Embodiment 455

The method of embodiment 453, wherein PCR is performed for unconvertedDNA where the unconverted DNA is a target for said method.

Embodiment 456

The method according to any one of embodiments 410-455, wheremethylation is determined for a promoter region of a gene selected fromthe group consisting of MGMT. RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4,RASGRF2, TM6SF1, and AKR1B1.

Embodiment 457

A method of detecting a cancer or the predisposition to a cancer in asubject, said method comprising:

providing a biological sample from said subject, wherein said biologicalsample comprises a DNA;

utilizing a set of cartridges according to any one of embodiments317-399, wherein said first cartridge of said set of cartridges is usedto perform a bisulfite conversion of said DNA; and said second cartridgeof said set of cartridges is used to desulphonate the converted DNA andto detect methylation of one or more gene promoters in said DNA whosemethylation state is a marker for a cancer, where an increase inmethylation of said one or more gene promoters is indicative of thepresence of a cancer or a predisposition to a cancer or a stage of acancer or precancer.

Embodiment 458

The method of embodiment 457, wherein said subject is a human.

Embodiment 459

The method according to any one of embodiments 457-458, wherein saidcancer is a cancer selected from the group consisting of breast cancer,pancreatic cancer, prostate cancer, brain cancer, a lung cancer, a Bcell lymphoma, a bronchus cancer, a colorectal cancer, a stomach cancer,an ovarian cancer, a urinary bladder cancer, a brain or central nervoussystem cancer, a peripheral nervous system cancer, an esophageal cancer,a cervical cancer, a melanoma, a uterine or endometrial cancer, a cancerof the oral cavity or pharynx, a liver cancer, a kidney cancer, abiliary tract cancer, a small bowel or appendix cancer, a salivary glandcancer, a thyroid gland cancer, a adrenal gland cancer, an osteosarcoma,a chondrosarcoma, a liposarcoma, a testes cancer, and a malignantfibrous histiocytoma.

Embodiment 460

The method according to any one of embodiments 457-458, wherein saidcancer is a cancer selected from the group consisting of breast cancer,pancreatic cancer, prostate cancer, brain cancer, a lung cancer.

Embodiment 461

The method according to any one of embodiments 457-460, wherein saidsample comprise a sample from serum or plasma.

Embodiment 462

The method according to any one of embodiments 457-460, wherein saidsample comprise an FFPE sample.

Embodiment 463

The method according to any one of embodiments 457-462, wherein said oneor more gene promoters comprise the promoters of one or more genesselected from the group consisting of RASSF1A, AKR1B1, HOXB4, HIST1H3C,RASGRF2, TM6SF1, BRCA1, BNC1, ADAMTS1, CDO1, SOX17, TAC1, HOXA7, andMGMT.

Embodiment 464

The method according to any one of embodiments 457-462, wherein saidcancer is pancreatic cancer and said one or more gene promoters comprisethe promoters of one, two, three, or four genes selected from the groupconsisting of ADAMTS1, and BNC1.

Embodiment 465

The method of embodiment 464, wherein said one or more gene promoterscomprise the promoter of ADAMTS1.

Embodiment 466

The method according to any one of embodiments 464-465, wherein said oneor more gene promoters comprise the promoter of BNC1.

Embodiment 467

The method according to any one of embodiments 457-462, wherein saidcancer is breast cancer and said one or more gene promoters comprise thepromoters of one, two, three, four, five, or all genes selected from thegroup consisting of BRCA1, RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2,and TM6SF1.

Embodiment 468

The method of embodiment 467, wherein said one or more gene promoterscomprise the promoter of BRCA1.

Embodiment 469

The method according to any one of embodiments 467-468, wherein said oneor more gene promoters comprise the promoter of RASSF1A.

Embodiment 470

The method according to any one of embodiments 467-469, wherein said oneor more gene promoters comprise the promoter of AKRIB1.

Embodiment 471

The method according to any one of embodiments 467-470, wherein said oneor more gene promoters comprise the promoter of HOXB4.

Embodiment 472

The method according to any one of embodiments 467-471, wherein said oneor more gene promoters comprise the promoter of HIST1H3C.

Embodiment 473

The method according to any one of embodiments 467-472, wherein said oneor more gene promoters comprise the promoter of RASGRF2.

Embodiment 474

The method according to any one of embodiments 467-473, wherein said oneor more gene promoters comprise the promoter of TM6SF1.

Embodiment 475

The method according to any one of embodiments 457-462, wherein saidcancer is breast cancer and said one or more gene promoters comprise thepromoter of BRCA1.

Embodiment 476

The method according to any one of embodiments 457-462, wherein saidcancer is lung cancer and said one or more gene promoters comprise thepromoters of one, two, three, for all genes selected from the groupconsisting of CDO1, SOX17, TAC1, and HOXA7.

Embodiment 477

The method of embodiment 476, wherein said one or more gene promoterscomprise the promoter of CDO1.

Embodiment 478

The method according to any one of embodiments 476-477, wherein said oneor more gene promoters comprise the promoter of SOX17.

Embodiment 479

The method according to any one of embodiments 476-478, wherein said oneor more gene promoters comprise the promoter of TAC1.

Embodiment 480

The method according to any one of embodiments 476-479, wherein said oneor more gene promoters comprise the promoter of HOXA7.

Embodiment 481

The method according to any one of embodiments 457-462, wherein saidcancer is brain cancer and said one or more gene promoters comprise thepromoter of MGMT.

Embodiment 482

A kit for the determination of DNA methylation, said kit comprising: acontainer containing a first cartridge and/or a second cartridge of setof cartridges according to any one of embodiments 317-399.

Embodiment 483

The kit of embodiment 482, wherein said first cartridge and said secondcartridge are contained in the same container.

Embodiment 484

The kit of embodiment 482, wherein said first cartridge and said secondcartridge are in separate containers.

Embodiment 485

The kit of embodiment 482, wherein said kit further comprises acontainer containing a lysis solution.

Embodiment 486

The kit of embodiment 485, wherein said lysis solution is a lysissolution for serum or plasma.

Embodiment 487

The kit of embodiments 485, wherein said lysis solution is a lysissolution for an FFPE sample.

Embodiment 488

The kit according to any one of embodiments 482-487, wherein said kitcomprises a container containing proteinase K.

Embodiment 489

The kit according to any one of embodiments 482-488, wherein said kitcomprises a conversion reagent in said cartridge or in a containerseparate from the cartridge.

Embodiment 490

The kit of embodiment 489, wherein said kit comprises said conversionreagent in a container separate from the cartridge.

Embodiment 491

The kit of embodiment 489, wherein said kit comprises said conversionreagent is provided in a chamber of the cartridge.

Embodiment 492

The according to any one of embodiments 489-491, wherein said conversionreagent comprises a compound selected from the group consisting ofsodium metabisulfite, potassium bisulfite, cesium bisulfite, ammoniumbisulfite, and DABSO.

Embodiment 493

The kit of embodiment 492, wherein said conversion reagent comprisesammonium bisulfite.

Embodiment 494

The kit according to any one of embodiments 482-493, wherein said kitcomprises a container containing a sample processing reagent.

Embodiment 495

The kit of embodiment 494, wherein said sample processing reagentcomprises guanidium thiocyanate.

Embodiment 496

The kit according to any one of embodiments 494-495, wherein said sampleprocessing reagent comprise ethanol.

Embodiment 497

The kit according to any one of embodiments 482-496, wherein said kitcontains instructional materials teaching the use of said cartridge forthe determination of DNA methylation.

In certain embodiments the methods and/or cartridges expressly excludemagnetic materials including magnetic glass, magnetic hydroxyapatite,and magnetic matrix materials. In certain embodiments the methods and/orcartridges expressly exclude magnetic materials for DNA isolation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates major components of a cartridge (e.g., a GENEXPERT®cartridge) suitable for use with the methods described herein. FIG. 1Bshows a top view of the cartridge illustrating chambers disposed arounda central valve.

FIG. 1C shows an illustrative workflow for the determination of DNAmethylation utilizing the reaction cartridge.

FIG. 2, panels A-C, illustrates one embodiment of a GENEXPERT® cartridgesuitable for the determination of DNA methylation as described herein.

FIGS. 3A-3C show illustrative, but non-limiting embodiments of themodules, and systems (e.g., processing units) for the determination ofDNA methylation. FIG. 3A illustrates a module for operation of aGENEXPERT® cartridge. FIG. 3B illustrates some components of oneembodiment of a module for operation of a cartridge for the analysis ofDNA methylation. FIG. 3C illustrates a system (e.g., processing unit)incorporating a plurality of modules.

FIGS. 4A-4D illustrate various strategies for the use of MethyLightprotocols to detect/quantify DNA phosphorylation. FIG. 4A, modified fromEads et al. 92000) Nucleic Acids. Res., 28(8): e32) schematicallyillustrates MethyLight technology. DNA is modified by sodium bisulfatewhich generates methylation-dependent sequence differences, e.g., at CpGdinucleotides by converting unmethylated cytosine residues (locationsindicated by white circles) to uracil, while methylated cytosineresidues (locations indicated by black circles) are retained ascytosine. Fluorescence-based PCR is then performed with primers thateither overlap methylation sites or that do not overlap any methylationsites. Sequence discrimination can occur either at the level of the PCRamplification process (panel D) or at the level of the probehybridization process (panel B), or both (panel D). Sequencediscrimination at the PCR amplification level utilizes primers andprobes (panel D), or just primers (panel C), to overlap potentialmethylation sites (e.g., CpG dinucleotides). Only two (fully methylated(M) and fully unmethylated (U)) of the many theoretical methylationpermutations are shown. The MethyLight assay can also be designed suchthat sequence discrimination does not occur at the PCR amplificationlevel. If neither the primers nor the probe overlap sites of methylation(e.g., CpG dinucleotides) (panel A), then no methylation-dependentsequence discrimination occurs at the PCR amplification or probehybridization level. This reaction represents amplification of theconverted genomic DNA without bias to methylation status, which canserve as a control for the amount of input DNA. When just the probeoverlaps methylation sites (panel B), then sequence discrimination canoccur through probe hybridization. FIG. 4B illustrates a MethyLightapproach using a single, e.g., methylation-specific, probe (PR3) alongwith methylation specific forward (FW) and reverse (RV) primers. FIG. 4Cillustrates a MethyLight approach using multiple probes (PR1 . . . PR5)that each target different regions. FIG. 4D illustrates a MethyLightapproach using multiple probes (PR1 . . . PR5) that each target the sameregion, but provide signals for different methylation patterns.

FIG. 5 illustrates results from a representative GeneXpert run from 300ng of HGDNA showing an ACTB qPCR curve and an HMBS qPCR curve.

FIGS. 6A and 6B illustrate the results of a titration forbisulfite-converted ACTB using human genomic DNA (hgDNA) in a 15 cyclenested qPCR (FIG. 6A) and a 20 cycle nested qPCR (FIG. 6B).

FIGS. 7A-7C shows the result of 20 cycles of nested qPCR (in thecartridge) for six methylated targets (AKR1B1, HOXB4, TM6SF1, RAASGRF2,and RASSF1A). FIG. 7A shows the results for 25 ng of HSDNA or 5000 cellswithout bisulfite conversion. FIG. 7B shows the results of 20 cycles ofnested qPCR for the bisulfite converted methylated targets using DNAfrom MBA-453 cells. FIG. 7C shows the results of 20 cycles of nestedqPCR for the bisulfite converted methylated targets using DNA fromMBA-453 cells in a carrier (1 μg of SS and 10 ng of HS DNA). Falloutsoccur at around 25-50 copies or around 100 cells.

FIG. 8 illustrates the results of a determination of conversionefficiency. The conversion efficiency is about 66% (˜1 Ct) thedifference between unconverted HMBS and converted ACTB.

FIG. 9 illustrates the increase in specificity for converted DNAproduced by nested qPCR.

FIG. 10 illustrates the specificity of the methylation cartridge. Thereis no priming off of unconverted DNA (top panel) or unmethylated DNA(bottom panel) except for HIST1H3C.

FIG. 11 shows illustrative but non-limiting workflows for analysis ofmethylation using a cartridge (e.g., a GENEXPERT® cartridge). Topillustrates one work flow for analysis of DNA methylation in a serum orplasma sample. Bottom illustrates one work flow for analysis of DNAmethylation in a tissue section (e.g., frozen or formalin-fixed paraffinembedded (FFPE) section).

FIG. 12 illustrates the results for a FFPE cell button for converted ALU(left-most curve) and methylated RASSF1A (right-most curve).

FIG. 13A illustrates a cartridge layout and FIG. 13B illustrates a flowchart of the protocol used in Example 4.

FIG. 14 illustrates a run in which some samples contain bisulfitecontamination.

FIG. 15A illustrates the results of 1000 MBA-453 cells with bisulfiteconversion (with HOXB4 giving the greatest signal). Figure. 15Billustrates results of 25 ng of HS DNA control (with only HIST1H3Cshowing a detectable signal).

FIG. 16 illustrates the structure of DABSO(1,4-diazoniabicyclo[2.2.2]octane-1,4-disulfonate).

FIG. 17 illustrates one embodiment of a cfDNA sample preparationcartridge. The cartridge is effective for both DNA and RNA isolation.The cartridge provides three GTC-ethanol washes (GTC-ethanol washes aretypically 1.25M guanidinium thiocyanate, 25 mM Tris pH 7.0, 50%ethanol), a PEG200 rinse, and a 15 mM KOH elution.

FIG. 18 illustrates controls for cfDNA extraction.

FIG. 19A shows a comparison of cfDNA preparation using a samplepreparation cartridge as described herein compared to a standardtube-fill (i.e. tube-based kit) preparation (left-most curve). Thecartridge preparation yield is very comparable to that obtained using atube fill method (right-most curve). FIG. 19B shows a comparison of theamount of extracted DNA detected using a cartridge-based DNA cleanup ascompared to a standard tube-fill as a function of DNA amount. Thecartridge-based method is conservatively within 1 Ct of the tube-fillmethods and is believed to be closer at higher DNA concentration.

FIG. 20A illustrates one embodiment of a high-volume (e.g., up to 12 ml)sample preparation (HVSP) cartridge that can be used with a qPCRcartridge and/or with a methylation detection cartridge. FIG. 20Bschematically illustrates one variation of work flows in the HVSPcartridge when used in combination with a qPCR cartridge to perform amethylation analysis.

FIG. 21 illustrates the detection of HBMS or β-globin using a twocartridge cleanup using a high-volume sample preparation cartridge (see,e.g., FIG. 20) where the sample is transferred from the high volumecartridge to the PCR analysis cartridge compared to detection using asample applied to a single PCR analysis cartridge resulting in lesssample volume.

FIG. 22 illustrates the results of bisulfite conversion using multipleheating steps (bottom panel) compared to a single heating step (toppanel).

FIG. 23A illustrates the steps and labor time for a methylation analysisusing a standard Qiagen DNA purification kit combined with a Zymo DNAmethylation kit (right) compared to a methylation analysis using aMethylation analysis cartridge described herein. FIG. 23B shows acomparison of the results obtained using the two different protocols.

FIG. 24 shows a comparison of DNA conversion using DABSO as theconversion reagent compared to DNA conversion using the Zymo bisulfiteconversion reagent.

FIG. 25, panels A and B, illustrates sensitivity of detection ofmethylated DNA. Panel A shows a dilution series of methylated DNA (MGMT)(the curves run from the highest concentrations on the left to thelowest concentration on the right. Panel B illustrates the sensitivityof detection of methylated pancreatic cancer markers.

FIG. 26 illustrates the results for a reverse complement multiplex assayfor both strands (curves from highest to lowest fluorescence: toppanel—BNC1_2, BNC1_2, BG, ADAMTS1_1/ADAMTS1_2; bottom panel—BNC1_2,BNC1_2, ADAMTS1_1/ADAMTS1_2, BG, ADAMTS1_1/ADAMTS1_2).

FIG. 27A illustrates the detection of both methylated DNA and mutationsin the same cartridge. Top panel illustrates detection of methylated DNAand a Kras G12D mutation in one cartridge, while bottom panelillustrates detection of methylated DNA and wildtype Kras in onecartridge. FIG. 27B illustrates detection of methylated DNA andmutations I the same cartridge in two pancreatic cancer cell lines:PANC-1 cells (top panel) and MIA-PaCa cells (bottom panel).

FIG. 28 illustrates temperature optimization for multiplex methylationanalysis of ADAMTS1, and BNC1 of a forward strand (top) and a reversestrand (bottom) of bisulfate-converted DNA.

FIG. 29 illustrates the ability to multiplex the MSP primer and probesets for BNC1, ADAMTS1, and a control gene ACTB. Probes were combinedinto two sets based on preferred conditions.

FIG. 30 illustrates one set of primers and probes used for detection ofMGMT methylation. Internal fwd 22150 (SEQ ID NO: 266); External fwd22422 (SEQ ID NO: 263); Probe 22419 (SEQ ID NO: 268), Internal rev 22151(SEQ ID NO: 267); external rev 22423 (SEQ ID NO: 265); template (SEQ IDNO: 1).

FIG. 31 shows the results of a comparison between bisulfatepyrsequencing and a MGMT methylation cartridge for extracted DNA (top)and for an FFPET sample (bottom).

FIG. 32 illustrates BRCA1 primer and probe set optimization of ΔCtbetween methylated converted and unmethylated converted DNA.

FIG. 33 illustrates a one target assay for BRCA1 methylation tested withthe ACTB control gene. As shown, eight different cell lines were testedand the effect of adding NH₄ was compared.

FIG. 34 illustrates the results of a three target methylation assay forgenes whose methylation is associated with lung cancer (SOX17, CD01,TAC1) in a background of normal plasma and in three different lungcancer cell lines.

FIG. 35 shows the results of a two-cartridge methylation analysis ofBNC1 and ACTB.

FIG. 36 shows the results of bisulfite conversion analysis of normalurine samples.

FIG. 37 shows the results of methylation analysis of normal and cancersputum samples.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “detect”, “detecting” or “detection” maydescribe either the general act of discovering or discerning or thespecific observation of a detectably labeled composition.

As used herein, the term “detectably different” or “spectrallydistinguishable” refers to a set of labels (such as dyes/fluorophores)that can be detected and distinguished simultaneously.

DNA methylation DNA methylation refers to the addition of a methyl group(CH₃) covalently to the base cytosine (C) typically in the dinucleotide5″-CpG-3″. The term CpG refers to the base cytosine (C) linked by aphosphate bond to the base guanine (G) in the DNA nucleotide sequence.

The term “conversion reagent” refers to a reagent that deaminatescytosine to uracil in single stranded DNA, while leaving 5-MeCessentially unaffected. Illustrative conversion reagents includebisulfates (e.g., sodium metabisulfite, potassium bisulfite, cesiumbisulfite, ammonium bisulfite, etc.) and/or compounds that can produce abisulfite under appropriate reaction conditions (e.g., DABSO).

The phrase “detecting methylation of a gene” generally refers to thedetection of methylation of cytosine, typically in CPG islands, in thepromoter region of the gene.

As used herein, the terms “patient” and “subject” are typically usedinterchangeably to refer to a human. In some embodiments, the methodsdescribed herein may be used on samples from non-human animals, e.g., anon-human primate, canine, equine, feline, porcine, bovine, lagomorph,and the like.

As used herein, the terms “oligonucleotide,” “polynucleotide,” “nucleicacid molecule,” and the like, refer to nucleic acid-containingmolecules, including but not limited to, DNA. The terms encompasssequences that include any of the known base analogs of DNA and RNAincluding, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “oligonucleotide,” refers to a single-strandedpolynucleotide typically having fewer than 500 nucleotides. In someembodiments, an oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to100, 12 to 75, or 12 to 50 nucleotides long. Oligonucleotides may bereferred to by their length, for example, a 24 residue oligonucleotidemay be referred to as a “24-mer.”

As used herein, the term “complementary” to a target gene (or targetregion thereof), and the percentage of “complementarity” of the probesequence to the target gene sequence is the percentage “identity” to thesequence of target gene or to the complement of the sequence of thetarget gene. In determining the degree of “complementarity” betweenprobes used in the compositions described herein (or regions thereof)and a target gene, such as those disclosed herein, the degree of“complementarity” is expressed as the percentage identity between thesequence of the probe (or region thereof) and sequence of the targetgene or the complement of the sequence of the target gene that bestaligns therewith. The percentage is calculated by counting the number ofaligned bases that are identical as between the 2 sequences, dividing bythe total number of contiguous nucleotides in the probe, and multiplyingby 100. When the term “complementary” is used, the subjectoligonucleotide is at least 90% complementary to the target molecule,unless indicated otherwise. In some embodiments, the subjectoligonucleotide is at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% complementary to the target molecule.

A “primer” or “probe” as used herein, refers to an oligonucleotide thatcomprises a region that is complementary to a sequence of at least 8contiguous nucleotides of a target nucleic acid molecule, such as atarget gene. In some embodiments, a primer or probe comprises a regionthat is complementary to a sequence of at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 29, at least 30, at least 319, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, or at least 40 contiguous nucleotides of a targetmolecule. When a primer or probe comprises a region that is“complementary to at least x contiguous nucleotides of a targetmolecule,” the primer or probe is at least 95% complementary to at leastx contiguous nucleotides of the target molecule. In some embodiments,the primer or probe is at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to the target molecule.

The term “nucleic acid amplification,” encompasses any means by which atleast a part of at least one target nucleic acid is reproduced,typically in a template-dependent manner, including without limitation,a broad range of techniques for amplifying nucleic acid sequences,either linearly or exponentially. Exemplary means for performing anamplifying step include polymerase chain reaction (PCR), ligase chainreaction (LCR), ligase detection reaction (LDR), multiplexligation-dependent probe amplification (MLPA), ligation followed byQ-replicase amplification, primer extension, strand displacementamplification (SDA), hyperbranched strand displacement amplification,multiple displacement amplification (MDA), nucleic acid strand-basedamplification (NASBA), two-step multiplexed amplifications, rollingcircle amplification (RCA), and the like, including multiplex versionsand combinations thereof, for example but not limited to, OLA/PCR,PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known ascombined chain reaction—CCR), digital amplification, and the like.Descriptions of such techniques can be found in, among other sources,Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., ColdSpring Harbor Press (1995); The Electronic Protocol Book, ChangBioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); TheNucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa,N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.;4(1):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451, Barany etal., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No.WO 01/92579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich etal., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide toMethods and Applications, Academic Press (1990); Favis et al., NatureBiotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102(2000); Belgrader, Barany, and Lubin, Development of a MultiplexLigation Detection Reaction DNA Typing Assay, Sixth InternationalSymposium on Human Identification, 1995 (available on the world wide webat: promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR KitInstruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002;Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook,Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res.27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66(2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl.Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002);Lage et al., Genome Res. 2003 Feb.; 13(2):294-307, and Landegren et al.,Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002Nov.; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 Feb.;12(1):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat.No. 5,686,243, PCT Publication No. W00056927A3, and PCT Publication No.W09803673A1.

In some embodiments, amplification comprises at least one cycle of thesequential procedures of: annealing at least one primer withcomplementary or substantially complementary sequences in at least onetarget nucleic acid; synthesizing at least one strand of nucleotides ina template-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated. Amplification can comprise thermocycling or, incertain embodiments, can be performed isothermally.

The term “hybridize” is typically used herein refer to “specifichybridization” which is the binding, duplexing, or hybridizing of anucleic acid molecule preferentially to a particular nucleotidesequence, in some embodiments, under stringent conditions. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target sequence, and to a lesser extentto, or not at all to, other sequences. A “stringent hybridization” and“stringent hybridization wash conditions” in the context of nucleic acidhybridization are sequence-dependent and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I, Ch. 2, “Overview of principles of hybridization and thestrategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”).Generally, highly stringent hybridization and wash conditions for filterhybridizations are selected to be about 5° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. In certain embodiments very stringentconditions are selected to be equal to the T_(m) for a particular probe.Dependency of hybridization stringency on buffer composition,temperature, and probe length are well known to those of skill in theart (see, e.g., Sambrook and Russell (2001) Molecular Cloning: ALaboratory Manual (3rd ed.) Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor Press, NY).

A “sample,” as used herein, generally refers to a biological sampleincluding biological fluids (e.g., blood or blood fractions, serum,plasma, pancreatic juice, cerebrospinal fluid, oral fluid, lymph,intraocular fluid, and the like) and/or tissue samples including, butnot limited to biopsy samples, frozen tissue samples, formalin fixedparaffin embedded (FFPE) samples from various tissues including but notlimited to breast tissue, endocervical tissue, vaginal tissue,colon/rectal tissue, throat tissue, and other types of human samples,such as blood, stool, and biopsy samples. The term sample also includesdiluted and/or buffered forms of the above samples, for example, abuffer into which a swab sample has been placed, a urine sample to whicha buffer has been added, and the like.

As used herein, the phrase “is indicative of the presence of a cancer ora predisposition to a cancer” means that a particular result tends toindicate that a cancer is present, and/or a precancerous condition ispresent or likely present. This phrase does not imply a definitivedetermination that the condition is present. A definitive determinationcan be made based on further examination or testing that a medicalpractitioner deems appropriate. Furthermore, this phrase does notrequire that a determination be made as to which condition may bepresent based only on the particular result. Rather, it is contemplatedthat a positive result will be considered in light of other examinationor text results to arrive at a differential diagnosis.

The term “tubefill procedure” refers to a procedure that is run usingstandard laboratory instrumentation rather than on a cassette (e.g.,rather than with a GENEXPERT®, or modified GENEXPERT® cartridgedescribed herein).

DETAILED DESCRIPTION

In various embodiments devices and methods are provided that facilitatethe rapid detection and/or characterization of methylation in DNAsamples. In certain embodiments automated reaction cartridges areprovided as are methods that that utilize the automated reactioncartridge(s) to facilitate analysis of the methylation of a DNA sampleand, optionally, to measure mRNA levels along with the determination ofDNA methylation. In various embodiments the DNA methylation isdetermined by bisulfite conversion and analysis of the bisulfiteconverted DNA (e.g., via methylation specific PCR, nucleic acidsequencing, melting point analysis, and the like). In certainembodiments the cartridge performs all or a part of the bisulfiteconversion of DNA and all or a part of the analysis of the bisulfiteconverted DNA. In certain embodiments the cartridge performs all of thesteps involved in bisulfite conversion and all or a part of the analysisof the bisulfite-converted DNA. In certain embodiments the cartridgeperforms all of the steps involved in bisulfite conversion and all ofthe analysis of the bisulfite-converted DNA. In certain embodiments thecartridge additionally performs an isolation and purification of the DNAto be analyzed.

There are several advantages to automating the methylation analysisincluding for example, reduction in overall processing time,improvements in efficiency, decreased user error and variability,minimization of loss between steps, and an improved ability to usesmaller amounts of sample. Use of a cartridge-based process, asdescribed herein, allows for rapid and easy testing of not only multiplesample types but also for evaluating methylation changes observed inseveral different types of cancers including, but not limited to breastcancer, colorectal cancer, prostate cancer, and lung cancer.

The cartridge-based methods described herein additionally permitmeasurement of mRNA derived from the same sample. Measurement ofcorresponding upstream and/or downstream mRNA involved in DNAmethylation can be important to understand the mechanism and activity ofthe epigenetic modification. For example, the measurement of DNAmethyltransferases (DNMT) mRNA has been studied along with DNAmethylation for several cancers (see Table 1).

TABLE 1 Illustrative DNA methyltransferases and their importance inparticular cancers (from Subramaniam et al. (2014) Front Oncol., 4:Article 80, doi: 10.3389/fonc.2014.00080). Methyl- transferase CancersDNMT1 Leukemia: upregulated - 5.3-fold expression Gastric cancer - 64.8%localized in the cytoplasm and nuclei Breast cancer - 16.6%Hepatocellular carcinoma - 100% Pancreatic cancer - highly expressed -Gli target gene Colon cancer - highly expressed Glioblastoma -overexpressed DNMT2 or Hepatocellular carcinoma - reduced expressionTRDMT1 Colorectal and stomach cancers - lower mRNA expression DNMT3AAcute myeloid leukemia - 22.1% mutations and affect translation Gastriccancer - 70.4% localized in the cytoplasm Breast cancer - 14%Hepatocellular carcinoma - 60% Pancreatic cancer - highly expressed -regulated by Gli 1 Colon cancer - highly expressed DNMT3B Leukemia:upregulated- 11.7-fold expression Gastric cancer - 51.9% localized inthe cytoplasm Breast cancer - 81.8% poor prognosis Breast cancer celllines-hypermethylation defect resulted in aberrant - overexpression DNMTactivity Hepatocellular carcinoma (60%) and mRNA levels high Coloncancer - highly expressed Prostate cancer - overexpressed Glioblastoma -overexpressed DNMT3L Cervical cancer - promising biomarker Embryonalcarcinoma - novel biomarker

Often separate independent extractions for DNA or RNA are used forstudying and measuring genes and transcripts. Co-detection from the samesample preparation would be ideal to minimize sample preparation, assayto assay, sample-to-sample and cell-to-cell variability.

Cartridge-Based Bisulfite Conversion of DNA

In certain embodiments the extraction of DNA, bisulfite conversion, andmethylation specific PCR are all performed in the cartridge. In oneillustrative embodiment, the user will add the sample to a lysis/bindingreagent, then mix/vortex the reagent briefly, and then add the sample toa sample port or chamber in the cartridge. Illustrative, butnon-limiting lysis reagents (including reagents particularly well suitedfor FFPE sections) are described in PCT Patent Publication No:WO/2014/052551 (PCT/US2013/061863), which is incorporated herein byreference for the reagents described therein.

Additional illustrative lysis reagents for serum or plasma and forformalin-fixed paraffin embedded (FFPE) samples are shown in Example(Tables 13, and 14, respectively).

In certain embodiments the cartridge is placed into a processing moduleand the assay is initiated by clicking through a set of selectionswithin software controlling the processing module (see, e.g., FIGS. 11Aand 11B). The cartridge then performs the bisulfite conversion processand analysis of the bisulfite-converted DNA. In certain embodiments mRNAis also determined. While in certain embodiments, all of the operationsare performed in the cartridge, in other embodiments, subsets of thevarious operations are performed in the cartridge as described below.

The sample can comprise any biological sample that contains DNA whosemethylation state is to be evaluated. Illustrative samples include, butare not limited to isolated DNA and/or isolated total nucleic acids, acell, a tissue, a biological fluid containing a nucleic acid, and thelike. In certain embodiments the biological sample comprises abiological fluid selected from the group consisting of plasma, serum,amniotic fluid saliva, mucus, urine, pancreatic juice, and cerebrospinalfluid. In certain embodiments the sample comprises a tissue sample froma healthy tissue, or a tissue sample from a diseased sample. In certainembodiments the tissue sample is from a fetus, a neonate, a child, anadolescent, or an adult. In certain embodiments the tissue samplecomprises tumor cell and/or is derived from a biopsy of a tumor (e.g., abreast cancer, a prostate cancer, a brain cancer, a cervical cancer, anovarian cancer, a pancreatic cancer, a colon cancer, a gastric cancer, ahepatocellular cancer and the like. In certain embodiments the samplecomprises a fixed tissue, e.g., a formalin fixed tissue sample. Incertain embodiments the sample comprises an embedded tissue sample(e.g., a formalin-fixed paraffin embedded (FFPE) tissue sample).

Bisulfite conversion of DNA typically involves four steps:

1) DNA purification;

2) DNA denaturation;

3) DNA conversion (e.g., bisulfite deamination); and

4) Alkali desulphonation.

Typically DNA conversion (e.g., using a conversion reagent such as abisulfite) involves: 1) Sulphonation: The addition of bisulphite to the5-6 double bond of cytosine; and 2) Hydrolic Deamination: hydrolyticdeamination of the resulting cytosine-bisulphite derivative to give auracil-bisulphite derivative. This is followed by Alkali Desulphonation:Removal of the sulphonate group by an alkali treatment, to give uracilas indicated above.

As noted above, in certain embodiments, the DNA purification can beperformed prior to placing a sample in the cartridge, or alternatively,can be performed by the cartridge itself. Accordingly, in certainembodiments the sample is added directly to the reaction cartridge,while in other embodiments, the sample is mixed with one or morereagents. In certain embodiments DNA preparation typically involvespreparing substantially isolated DNA. This may involve lysing cells torelease DNA, removing particulates and cellular debris, and/or removingprotein components to provide a sample comprising substantially purenucleic acids (e.g., substantially pure DNA and/or a substantially purecombination of DNA and RNA). In one illustrative, but non-limiting,embodiment, the sample (e.g., a tissue sample) is added to a lysisreagent, agitated and then inserted into the cartridge for furtherprocessing.

In certain embodiments, all of the reagents necessary to performbisulfite conversion of the DNA are provided in the cartridge. Incertain embodiments, to avoid degradation of reagents over time in thecartridge, certain reagents may be added to the cartridge immediatelybefore use. Thus, for example in certain embodiments, it is contemplatedthat the cartridge may be loaded with a conversion reagent (e.g., abisulfite reagent) and/or a guanidium thiocynanate reagent (e.g.,GTC-EtOH-Tween) at or about the time the sample is loaded into thecartridge. In certain embodiments, the guanidinium thiocyanate reagent(e.g., GTC-EtOH-Tween) is combined with the sample and added to thecartridge in the sample receiving chamber (e.g., chamber 2 in theGENEXPERT® cartridge).

In certain embodiments when performing the bisulfite conversion of DNAusing a reaction cartridge (e.g., GENEXPERT® cartridge), the methodcomprises

i) contacting a biological sample comprising a nucleic acid to a firstmatrix material comprising a first column or filter where said matrixmaterial binds and/or filters nucleic acids in said sample and therebypurifies the DNA;

ii) eluting the bound DNA from the first matrix material (e.g., using analkaline solution) and denaturing the DNA to produce eluted denaturedDNA;

iii) heating the eluted DNA in the presence of a conversion reagent(e.g., a reagent that provides bisulfite ions) to produce a converted(e.g., a deaminated) nucleic acid;

iv) contacting the converted nucleic acid to a second matrix materialcomprising a second column to bind said deaminated nucleic acid to saidsecond matrix material (note in certain embodiments the second columncan be a column different than the first column, or in otherembodiments, the same column used a second time);

v) desulphonating the bound deaminated nucleic acid and/orsimultaneously eluting and desulphonating the nucleic acid by contactingthe deaminated nucleic acid with an alkaline solution to produce aconverted (e.g., bisulfite converted) nucleic acid; and

vi) eluting the converted nucleic acid from said second matrix material,wherein at least steps iv) through vi) are performed in a one reactioncartridge.

In certain embodiments the method further includes the analysis of theconverted DNA. Accordingly, in certain embodiments, the method furthercomprises:

vii) performing methylation specific PCR and/or nucleic acid sequencing,and/or high resolution melting analysis (HRM) on the converted nucleicacid to determine the methylation of the nucleic acid, wherein at leaststeps iv) through vi) are performed in a single reaction cartridge.

In certain embodiments at least steps iii) through vi) are performed inone reaction cartridge.

In certain embodiments at least steps ii) through vi) are performed inone reaction cartridge.

In certain embodiments at least steps i) through vi) are performed inone reaction cartridge.

In certain embodiments at least steps i) through vii) are performed inone reaction cartridge.

It is noted that the first column and, where present, the second columncan refer to discrete columns. However, particularly when integratedinto a reaction cartridge, the “column” can simply be a matrix materialdisposed in a chamber or channel in the cartridge. In variousembodiments the “columns” act as filters and/or as affinity columns thatbind nucleic acids. Accordingly, in certain embodiments the columncontains a matrix material that binds a nucleic acid (e.g., DNA and/orRNA). Illustrative matrix materials include, but are not limited to,glass (silica), an ion exchange resin, hydroxyapatite, and the like. Itwill be recognized that the matrix materials can take a number of forms.Thus, in certain embodiments, the matrix material comprises a fibrousmaterial a particulate material (e.g., microbeads, nanobeads, etc.), astructured material (e.g., porous “baffle” system”, a serpentinechannel, and the like). In certain embodiments the first column andsecond column are different columns (chambers or channels). In otherembodiments the first column and the second column are the same column(chamber or channel) that is used twice (e.g., a first time and a secondtime).

In certain embodiments, the use of one or more additional filters, e.g.,to clean up the initial sample prior to contacting with the first matrixmaterial, is contemplated. Thus, for example, in certain embodiments, afilter matrix (e.g., polycarbonate filter, nylon filter, polypropylenefilter, polyester filter, nylon filter, ceramic filter,polytetrafluoroethylene filter, and the like) is disposed in the samplereceiving chamber or “downstream” from the sample receiving chamber andbefore the first “column”. It is also recognized, that in certainembodiments, the sample, can be lysed and/or filtered prior todeposition into a sample receiving chamber.

In certain illustrative, but non-limiting embodiments, the methodsdescribed herein can be performed using a GENEXPERT® cartridge (Cepheid,Inc., Sunnyvale, Calif.) or a variant thereof. In various embodimentssample extraction, and/or amplification, and/or DNA conversion, and/ordetection can all be carried out within this self-contained “laboratoryin a cartridge” (see, e.g., U.S. Pat. Nos. 5,958,349, 6,403,037,6,440,725, 6,783,736, and 6,818,185, each of which is hereinincorporated by reference in its entirety). In various embodimentscomponents of the cartridge can include, but are not limited to,processing chambers containing reagents, filters, and capturetechnologies useful to extract, purify, and amplify target nucleicacids. A valve enables fluid transfer from chamber to chamber andcontains nucleic acids lysis and filtration components. An opticalwindow enables real-time optical detection (e.g., of PCR amplificationproducts). A reaction tube can be provided that permits very rapidheating and/or thermal cycling.

In certain embodiments an illustrative GENEXPERT® cartridge comprises aplurality of chambers disposed around a central valve assembly andselectively in fluid communication with the central valve assembly wherethe central valve assembly is configured to accommodate a plunger thatis capable of drawing fluid into or out of a chamber in fluidcommunication with the central valve. Rotation of the valve assemblydetermines which chamber are in fluid communication with the centralvalve. One illustrative GENEXPERT® cartridge is illustrated in FIG. 1Awhich show the cartridge, processing/reagent chambers, a reaction tube(e.g., heating and/or thermocycling tube), optional optical windows, anda valve that facilitates fluid transfer from chamber to chamber.

An illustrative layout of the cartridge is shown in FIG. 1B whichprovides a top view of the cartridge identifying various chambers bynumber. In one illustrative, but non-limiting embodiment, the componentsof the chambers comprising the cartridge are as listed in Table 2. Itwill be recognized that this disposition of reagents and chamber isillustrative and non-limiting. Using the teachings provided herein otherreagent dispositions and/or other chamber configurations will beavailable to one of skill in the art.

TABLE 2 One illustrative embodiment showing chamber contents for use ofa GENEXPERT ® cartridge for measurement of DNA methylation. InitialVolume Chamber # Chamber Contents (μL) 1 —  2* Sample chamber (samplemixed with e.g., GTC-EtOH-Tween precipitation reagent)  3** GTC-EtOH  4*** Bisulfite reagent (e.g., 8M ammonium bisulfite) 5 Buffer e.g., 50mM Tris pH 8.5 6 — 7 — 8 Rinse (e.g., PEG 200) 9 Beads (e.g.,polymerase, primer, probe) 10  Elution/Desulphonation reagent (e.g., 15mM KOH) 11  Beads (e.g., polymerase, primer, probe) *Sample is added tochamber 2 by user **In certain embodiments, GTC-EtOH is added at time ofuse (e.g., when sample is added). In certain embodiments GTC-EtOH isprovided as reagent already disposed in cartridge. ***In certainembodiments, bisulfite reagent is added at time of use (e.g., whensample is added). In certain embodiments bisulfite reagent is alreadydisposed in cartridge.

One embodiment of a step-by-step workflow for the determination of DNAmethylation utilizing such a cartridge is shown in FIG. 1C. In thiscartridge configuration, there are five chambers that, in use (e.g.,when the cartridge is operating to determine DNA methylation), will holdreagents and buffers (e.g., chambers 3, 4, 5, 8, and 10), one chamberthat will hold the sample added by the user (e.g., chamber 2), and oneor two (or more) chambers holding analysis reagents (e.g., MSP reagentssuch as enzyme reaction, template specific reaction, and/or or 200 mMTris pH 7.0, e.g., as beads) (e.g., chambers 9, and 11). In certainembodiments, the reagents (e.g., polymerase, reverse transcriptase,primer, probe) are provided in solution. In certain embodiments thereagents are provided as lyophilized powders. In certain embodiments thereagents are provided as lyophilized beads. The beads can furthercomprise agents to that improve reagent stability and/or activity (see,e.g., U.S. Patent Publication No: 2006/0068399 which is incorporatedherein by reference for the beads, bead fabrication, and beadformulations described therein.

In certain embodiments the cartridge, as provided contains all of thereagents necessary to run the cartridge and only the sample (e.g.,sample in buffer/lysis/precipitation solution) is added to thecartridge. In certain embodiments the cartridge is provided without theGTC-EtOH and/or the bisulfite reagents and one or both are added at thetime of use. Thus, in certain embodiments, the GTC-EtOH reagent is addedto the cartridge at the time of use, in certain embodiments thebisulfite reagent (in addition to the sample) is added to the chamber atthe time of use, and in certain embodiments, both the GTC-EtOH and thebisulfite reagent (in addition to the sample) are added to the cartridgeat the time of use. In certain embodiments these reagents are addeddirectly to the desired chambers (see, e.g., Table 2). In certainembodiments ports are provided for loading the reagents and the portsare configured to deliver the reagent(s) to the desired chambers.

At the start of the assay, the cartridge dispenses the sample, e.g. fromchamber 2 over a glass fiber column (e.g. the first column) in thecartridge. DNA is eluted off the column and simultaneously denatured byan alkali solution, e.g., a low concentration of potassium hydroxidefrom chamber 10 into a concentrated bisulfite reagent (e.g.,concentrated ammonium bisulfite) in Chamber 4. In certain embodimentsthe DNA is eluted with an alkaline solution of KOH with a pH greaterthan about 10.5, or a pH greater than about pH 12. In certainembodiments the DNA is eluted with 10-15 mM KOH.

As indicated above, the DNA is eluted (optionally with a burst ofsonication) into the bisulfite reagent. In various embodiments theconversion reagent (e.g., bisulfite reagent) is present at aconcentration ranging from about 4 M to about 10 M, or from about 5 M toabout 8 M, or from about 6 M or about 7 M. In certain embodiments thebisulfite solution comprises sodium metabisulfite, or potassiumbisulfite, or ammonium bisulfite, or cesium bisulfite, or DABSO(1,4-diazoniabicyclo[2.2.2]octane-1,4-disulphinate, see, e.g., FIG. 16).In certain embodiments the conversion reagent (e.g., bisulfite reagent)contains radical scavengers, including, but not limited to one or morechemicals to prevent sulfite oxidation to sulfate (TROLOX andhydroquinone), and/or catalysts (polyamines).

The DNA-bisulfite (DNA/conversion reagent) mix is then introduced into atemperature controlled chamber or channel and incubated at a temperatureranging from about 40° C. to about 95° C. In certain embodiments the mixis incubated at a constant temperature, while in other embodiments,e.g., where the temperature controlled chamber or channel is athermocycling chamber or channel (e.g., a smartcycler tube in the backof the cartridge), the mix is thermally cycled (e.g., between 60° C. and95° C.). The mix is incubated until the DNA is converted (e.g.,deaminated). In certain embodiments the incubation is for a period oftime that ranges from about 5 minutes up to about 4 hours, or preferablyfrom about 15 minutes up to about 45 minutes.

Following incubation the DNA/conversion reagent) (e.g., DNA-bisulfite)solution is mixed with fresh guanidinium thiocyanate-EtOH, e.g., fromchamber 3 and dispensed over a matrix material. In certain embodimentsthe first column is reused, hence there is only one column and thesecond column and the first column are the same. In certain embodimentsthe second column is a separate column different than the first column.

The DNA bound to the second column matrix material is washed with freshGTC-EtOH (e.g., from chamber 3) and rinsed (e.g., with a PEG 200 rinse,e.g., from chamber 8). The DNA is then desulphonated on the column, oris simultaneously eluted and desulphonated by contacting the deaminatednucleic acid with an alkaline solution (e.g., KOH from chamber 10 toproduce a bisulfite converted nucleic acid. In certain embodiments theincubation is for a period of time ranging from about 1 minute to about1 hour, or from about 5 minutes to about 30 minutes, or from about 10minutes to about 20 minutes, or for about 15 minutes.

Where the initial incubation was in a thermocycling chamber that is tobe further used, the thermocycling chamber or channel is washed with abuffer to remove residual bisulfite and neutralize pH. It was asurprising discovery that incubation with a conversion reagent (e.g., abisulfite reagent), and/or desulphonation can be performed in a channelor chamber that is later used for PCR without bisulfite contaminationsubstantially interfering with the later PCR reaction(s).

The eluted desulphonated bisulfite-converted DNA can be mixed with anappropriate buffer and analyzed for methylation. In certain embodimentsthe converted DNA is mixed with concentrated Tris, enzyme reaction, andtemplate specific beads (e.g., beads comprising primers and/or probesfor the PCR or nested PCR reaction(s)) in chambers 9 and 11, and thefinal mixture is aspirated into the thermocycling tube or chamber forthe methylation specific quantitative PCR reaction.

Bisulfite contamination during the qPCR step can be the primary failuremode of the methylation cartridge. Residual bisulfite can result fromeither direct contamination of the PCR reaction tube (e.g., during thebisulfite conversion step) or from indirect contamination (e.g. crosscontamination during bisulfite fluidic movements between chambers).Residual bisulfite contamination, if present, can be measured bybisulfite-mediated probe cleavage during the qPCR step, which results inan increase in fluorescence during the earlier qPCR cycles (cycles 1-10)typically used for background subtraction. Accordingly, in certainembodiments, the cartridge comprises beads that provide one or moreprobes that are cleavable during PCR if bisulfite is present. Results ofa run containing bisulfite contamination are shown in FIG. 14.

While the methods above (and in Example 4, see, e.g., FIG. 13A) aredescribed with respect to specific chambers in the GENEXPERT® cartridge,it will be recognized that the particular reagent/chamber assignmentscan be varied depending on the particularities of the methylationanalysis protocol applied.

Thus, for example, operation of a methylation analysis cartridge (e.g.,a GENEXPERT® cartridge can be generally described by a flow chart (see,e.g., FIGS. 1C and 13B). In the illustrative, but non-limitingembodiment shown in FIG. 13B, the DNA sample is provided in a bindingbuffer (e.g., a buffer comprising GTC-EtoH, in certain embodiments afterthe sample is processed with proteinase K and/or a lysis solution). Incertain embodiments the sample is obtained from a sample preparationcartridge as described herein (see, e.g., FIG. 20).

The sample in binding buffer is introduced into a sample receivingchamber of the cartridge. In operation the cartridge is operated todeliver the sample solution to a matrix (“column”) that binds the DNA.The bound DNA is then eluted from the column using an alkaline reagent(e.g., KOH solution) combined with a bisulfite reagent and moved to aheating tube (typically the PCR reaction tube) in the cartridge wherethe bisulfite reaction proceeds (e.g., at about 50° C. or about 60° C.to about 90° C. for about 45 minutes (or up to about 90 minutes), inthis illustrative protocol). The reacted DNA is combined with a bindingbuffer (e.g., 2.25 M Guanidinium thiocyanate, 22.5 mM Tris pH 7.0, 0.5%Tween20, 50% Ethanol, and 0.005% SE-15 antifoam (a 10% emulsion of anactive silicon antifoam and non-ionic emulsifiers)) and moved back tothe same column, or to a different column, where it again binds to thecolumn matrix. The reacted DNA is washed with GTC-EtOH, rinsed with PEG(e.g., PEG200) and eluted again from the column using an alkalinereagent (e.g., KOH) which also desulphonates the DNA. While the DNA isdesulphonating the reaction tube (e.g., PCR reaction tube) can be heatedand rinsed (e.g., 10× rinse) to remove any bisulfite reagent. The elutedDNA (or a portion thereof) can be moved to a reaction tube for PCRand/or nested PCR.

It will be appreciated that these operations can be performed on theentire sample or on a portion of the DNA sample. In the latter case aportion of the sample can be stored in one or more chambers and used asa control, or subjected to a different analysis/protocol.

Co-Purification and Detection of Both RNA Expression and DNAMethylation.

In certain embodiments methods for co-purification and detection of bothaltered RNA expression of genes along with DNA methylation (MSP) in acartridge-based assay (e.g., utilizing a GENEXPERT® cartridge) areprovided. In certain embodiments these assays would identify alteredexpression of e.g. DNMT correlated with tumor-specific methylation fromthe same sample preparation. In certain embodiments these assays can beused to verify expression and methylation status.

We have shown that we can elute nucleic acids off the column using aTris buffered elution that retains a portion of nucleic acids on thecolumn. In one illustrative embodiment, an RNA fraction is eluted andretained, e.g., in a chamber in the cartridge using a Tris solution.

After saving the RNA fraction, NaOH or KOH elution which will strip thecolumn and elute and denature the DNA which would go into bisulfite forconversion as described above. Then, ether using the RNA elutionfraction to elute the bisulfite converted DNA from the column or usingthe KOH elution mix the two fractions (RNA and converted DNA products)are mixed for RNA plus bisulfite converted qRT-PCR. This involvesincorporating a reverse transcriptase (RT) step for the RNA plus MSP (orother analytic method) in the same tube from the same sample.Alternative methods include, but are not limited to performing the RTstep independently prior to mixing with DNA (combine cDNA and DNA) forqPCR, or PCR for DNA or RT RNA could be done independently/seriallyusing one thermocycling tube/chamber or simultaneously using multiplethermocycling tubes/chambers in the cartridge.

Analysis of Converted DNA

Numerous analytic methods can be performed in the cartridge to evaluateDNA methylation. Alternatively, in certain embodiments, the cartridgecan be coupled to another device and/or system for further analysis ofthe converted (e.g., bisulfite or DABSO converted) DNA. Illustrativemethods include, but are not limited to methylation specific PCR (MSP),direct sequencing, high resolution melting analysis (HRM),pyrosequencing (sequencing by addition), base-specific cleavage analysis(e.g. base-specific MALDI-TOF), and the like.

Methylation-Specific PCR (MSP).

In various embodiments methylation-specific PCR can be used to evaluatemethylation status of the target DNA. MSP utilized primer and/or probesets designed to be “methylated-specific” by including sequencescomplementing only unconverted 5-methylcytosines, or, on the converse,“unmethylated-specific”, complementing thymines converted fromunmethylated cytosines. Methylation is then determined by the ability ofthe specific primer to achieve amplification. This method isparticularly effective for interrogating CpG islands in regions of highmethylation density, because increased numbers of unconvertedmethylcytosines within the target to be amplified increase thespecificity of the PCR. In certain embodiments placing the CpG pair atthe 3′-end of the primer also improves the specificity.

In certain embodiments methylation is evaluated using a MethyLightmethod. The MethyLight method is based on MSP, but provides aquantitative analysis using quantitative PCR (see, e.g., Eades et al.(2000) Nucleic Acids Res., 28(8): E32. doi:10.1093/nar/28.8.e32).Methylated-specific primers are used, and a methylated-specificfluorescence reporter probe is also used that anneals to the amplifiedregion. In alternative fashion, the primers or probe can be designedwithout methylation specificity if discrimination is needed between theCpG pairs within the involved sequences. Quantitation can be made inreference to a methylated reference DNA. One modification to thisprotocol to increase the specificity of the PCR for successfullybisulphite-converted DNA (ConLight-MSP) uses an additional probe tobisulphite-unconverted DNA to quantify this non-specific amplification(see, e.g., Rand et al. (2002) Methods 27(2): 114-120).

In various embodiments the MethyLight methods utilize TAQMAN®technology, which is based on the cleavage of a dual-labeled fluorogenichybridization probe by the 5′ nuclease activity of Taq-polymerase duringPCR amplification (Eads et al. (1999) Cancer Res., 59: 2302-2306; Livaket al. (1995) PCR Meth. Appl., 4: 357-362; Lee et al. (1993) NucleicAcids Res., 21: 3761-3766; Fink et al. (1998) Nat. Med., 4: 1329-1333).The use of three different oligonucleotides in the TAQMAN® technology(forward and reverse PCR primers and the fluorogenic hybridizationprobe) offers the opportunity for several sequence detection strategies.

For example, the sequence discrimination can occur at the level of thePCR amplification process (see, e.g., FIG. 4A, panel C) and/or at thelevel of the fluorogenic probe hybridization (see, e.g., FIG. 4A, panelB). In both steps, the discrimination is based on the differentialannealing of the perfectly matched, versus mismatched oligonucleotides.In the MethyLight technology, sequence discrimination at the PCRamplification level occurs by designing the primers and probe, or justprimers, or just probes, to overlap potential sites of DNA methylation(e.g., CpG dinucleotides). One approach is simply a fluorescence-basedversion of the MSP technique (Herman et al. (1996) Proc. Natl. Acad.Sci. USA, 93: 9821-9826). Each oligonucleotide (primers and probe) cancover anywhere from zero to multiple CpG dinucleotides. Each CpGdinucleotide can result in two different sequence variations followingbisulfite conversion, depending on whether that particular site wasmethylated (mCpG) or unmethylated (UpG). For example, if anoligonucleotide overlaps two CpG dinucleotides, then the number ofpossible sequence variants in the genomic DNA within the region coveredby that oligonucleotide is 2×2=4. If both of the primers and the probeeach overlap two CpGs, then the total number of variants containedwithin the sequence covered by the oligonucleotides is 4×4×4=64. Intheory, one could design separate PCR reactions to analyze the relativeamounts of each of these potential 64 sequence variants. However,significant methylation information can be derived from the analysis ofa much smaller number of variants by designing reactions for the fullymethylated and fully unmethylated molecules, which represent the twomost extreme sequence variants this hypothetical example. The ratiobetween these two reactions or the ratio between the methylated reactionand a control reaction provides a measure of the prevalence ofmethylated molecules at this locus.

The MethyLight technology can also be modified to avoid sequencediscrimination at the PCR amplification level. If the neither theprimers nor the probe overlie any CpG dinucleotides, then the reactionrepresents unbiased amplification and can serve as a control for theamount of input DNA. One illustrative useful control reaction is one inwhich the entire amplicon is devoid of any CpG dinucleotides in theunconverted genomic sequence. When just the probe is designed to coverCpG dinucleotides, then sequence discrimination occurs solely at thelevel of probe hybridization. In this version, all sequence variantsresulting from the sodium bisulfite conversion step are amplified withequal efficiency, as long as there is no amplification bias (see, e.g.,Wamecke et al. (1997) Nucleic Acids Res., 25: 4422-1426). In this case,the design of separate probes for each of the different sequencevariants associated with a particular methylation pattern (2×2=4 probesin the case of two CpGs) allows a quantitative determination of therelative prevalence of each sequence permutation in the mixed pool ofPCR products.

In certain embodiments the analysis methods also provide PCR specificfor unconverted DNA. This PCR may interrogate SNPs, mutations, and/ortranslocations, etc. In this regard, it is noted that the detection ofmutations and methylation in a single cartridge is illustrated inExample 12 (see, e.g., FIGS. 27A and 27B). Detection of SNPs, mutations,translocations and the like can readily be accomplished by the inclusionof primers and probe sets specific for the detection of these targets.

Nested PCR and Multiplex PCR Assays.

In certain embodiments methylated DNA can be detected using an PCRmethods well known to those of skill in the art. In certain embodimentsa nested PCR reaction is used to detect methylation targets. In oneillustrative, but non-limiting, embodiment (see, e.g., Example 4), anested PCR protocol can be used where the first 15-20 cycle PCR reactionis not specific for methylation but only the converted DNA sequences(i.e., they do not cross CpGs or in instances when they do a R=purine orY=pyrimidine is used to catch both methylated and unmethylated templatesequences). The second qPCR reaction (e.g., a 45 cycle qPCR reaction)can contain both primers and probes that are specific for typically 2-3methylated CpGs.

It will be noted that in certain embodiments, a MethyLight analysis isperformed using a single probe (see, e.g., FIG. 4B). In this approach,using a single, e.g., methylation-specific, probe (PR3) along withmethylation specific forward (FW) and reverse (RV) primers, methylationspecific PCR for the probe (PR3) provides a signal that is dependent onmethylation and bisulfite conversion for the FW, RV and PR3 sequences.

In various embodiments, multiplexed PCR assays are contemplated. By wayof illustration, FIG. 4C illustrates a MethyLight approach usingmultiple probes (PR1, PR2, . . . PR5) that each target differentregions. The combined signal from all the probes (PR1, PR2, PR3, PR4,and PR5) yields a measure of the amount/degree of methylation. Incertain embodiments each probe has its own specific dye/fluor so that itis detectable independently of the other probes. Thus, even where onetarget is not methylated, a signal may still be detected, e.g., if PR3is not methylated there will be no/less signal from the remainingprobes. FIG. 4D illustrates a MethyLight approach using multiple probes(PR1 . . . PR5) that each target the same region, but provide signalsfor different methylation patterns. While the approach illustrated inFIG. 4C can provide detection from a larger region, this multi-probeapproach on a single smaller region could be accomplished with sequencespecific primers or probes interrogating the extent of methylationacross a specific sequence after bisulfite conversion.

In certain embodiments a reverse complement multiplex assay for bothstrands can be used (see, e.g., FIG. 26). Following bisulfiteconversion, both strands lose their complementarity. Thus, primer andprobe sets can be designed for one strand or the other, and result inunique amplicons. In addition to providing “more opportunities” fordetection, this approach can potentially help with sensitivity (at LOD,if only one strand or the other ends up in the tube, this approach wouldensure the signal gets picked up). This approach permits the multiplexassay to be expanded to detect different CpGs at the same promoter site.The reverse complement multiplex provides more opportunities to detecttarget methylation and to pick up heterogenous methylation.

The foregoing methods are illustrative and non-limiting. Using theteachings provided herein numerous variations of MSP and/or MethyLightanalysis will be available to one of skill in the art and implementableon a reaction cartridge, e.g. as described herein.

Direct Sequencing

In certain embodiments methylation status of the DNA can be determinedusing direct sequencing methods. In certain embodiments, the method canutilize PCR and standard dideoxynucleotide DNA sequencing to directlydetermine the nucleotides resistant to bisulphite conversion (see, e.g.,Frommer et al. (1992) Proc. Natl. Acad. Sci. USA, 89 (5): 1827-1831). Invarious embodiments primers are designed to be strand-specific as wellas bisulphite-specific (e.g., primers containing non-CpG cytosines suchthat they are not complementary to non-bisulphite-treated DNA), flanking(but not involving) the methylation site of interest. Therefore, it willamplify both methylated and unmethylated sequences, in contrast tomethylation-specific PCR. All sites of unmethylated cytosines aredisplayed as thymines in the resulting amplified sequence of the sensestrand, and as adenines in the amplified antisense strand. In certainembodiments nested PCR methods can be used to enhance the product forsequencing.

In certain embodiments the sequencing can be performed in the cartridge.In other embodiments, the cartridge can be coupled (e.g., fluidiccoupled) to a sequencing machine to provide the sequencing analysis.Alternatively, in certain embodiments, the amplified product can bemanually transferred from the cartridge to the sequencing system.

High Resolution Melting Analysis (HRM)

In certain embodiments high-resolution melting analysis (HRM) can beused to differentiate converted from unconverted bisulphite-treated DNA.HRM is a quantitative PCR technique in which the PCR amplicons areanalyzed directly by temperature ramping and resulting liberation of anintercalating fluorescent dye during melting (see, e.g., Wojdacz andDobrovic (2007) Nucleic Acids Res. 35(6): e41). The degree ofmethylation, as represented by the C-to-T content in the amplicon,determines the rapidity of melting and consequent release of the dye.This method allows direct quantitation, but assesses methylation in theamplified region as a whole rather than at specific CpG sites.

Pyrosequencing

In certain embodiments pyrosequencing (sequencing by synthesis) can beused to analyze bisulphite-treated DNA without usingmethylation-specific PCR (see, e.g., Colella et al. (2003).BioTechniques 35(1): 146-150; Tost et al. (2003) BioTechniques 35(1):152-156; and the like). Sequencing by synthesis differs from Sangersequencing in that it utilizes the detection of phosphate release onnucleotide incorporation, rather than chain termination withdideoxynucleotides. The DNA sequence is able to be determined by lightemitted upon incorporation of the next complementary nucleotide by thefact that typically only one out of four of the possible A/T/C/Gnucleotides are added and available at a time so that only one lettercan be incorporated on the single stranded template (which is thesequence to be determined).

Following PCR amplification of the region of interest, pyrosequencingcan be used to determine the bisulphite-converted sequence of specificregions (e.g., CpG sites). In certain embodiments the ratio of C-to-T atindividual sites can be determined quantitatively based on the amount ofC and T incorporation during the sequence extension.

A modification of this technique can utilize allele-specific primersthat incorporate single-nucleotide polymorphisms (SNPs) into thesequence of the sequencing primer(s), thus allowing for separateanalysis of maternal and paternal alleles (see, e.g., Wong et al. (2006)BioTechniques 41(6): 734-739). This modification is particularly of usefor genomic imprinting analysis.

Base-Specific Cleavage Analysis.

In certain embodiments, base-specific cleavage/MALDI-TOF takes advantageof bisulphite-conversions by adding a base-specific cleavage step toenhance the information gained from the nucleotide changes (Ehrich etal. (2005) Proc. Natl. Acad. Sci. USA, 102 (44): 15785-15790). By firstusing in vitro transcription of the region of interest into RNA (byadding an RNA polymerase promoter site to the PCR primer in the initialamplification), RNase A can be used to cleave the RNA transcript atbase-specific sites. RNase A cleaves RNA specifically at cytosine anduracil ribonucleotides and base-specificity is achieved by addingincorporating cleavage-resistant dTTP when cytosine-specific(C-specific) cleavage is desired, and incorporating dCTP whenuracil-specific (U-specific) cleavage is desired. The cleaved fragmentscan then be analyzed by MALDI-TOF or other methods. Bisulphite treatmentresults in either introduction/removal of cleavage sites by C-to-Uconversions or shift in fragment mass by G-to-A conversions in theamplified reverse strand. C-specific cleavage will cut specifically atall methylated CpG sites. By analyzing the sizes of the resultingfragments (e.g., using MALDI-TOF, capillary electrophoresis, microchipelectrophoresis, and the like), it is possible to determine the specificpattern of DNA methylation of CpG sites within the region, rather thandetermining the extent of methylation of the region as a whole.

Methylation-Sensitive Single-Strand Conformation Analysis (MS-SSCA).

Methylation-sensitive single strand conformation analysis (MS-SSCA) isbased on the single-strand conformation polymorphism analysis (SSCA)method developed for single-nucleotide polymorphism (SNP) analysis(Bianco et al. (1999) Hum. Mutat. 14(4): 289-293). SSCA differentiatesbetween single-stranded DNA fragments of identical size but distinctsequence based on differential migration in non-denaturatingelectrophoresis. In MS-SSCA, this is used to distinguish betweenbisulphite-treated, PCR-amplified regions containing the CpG sites ofinterest. Although SSCA lacks sensitivity when only a single nucleotidedifference is present, bisulphite treatment frequently makes a number ofC-to-T conversions in most regions of interest, and the resultingsensitivity can be high. In certain embodiments MS-SSCA can also providesemi-quantitative analysis of the degree of DNA methylation based on theratio of band intensities. Typically, however, MS-SSCA assesses all CpGsites as a whole in the region of interest rather than individualmethylation sites.

Methylation Targets.

As noted above, DNA methylation is of interest in a wide number ofcontexts. In certain embodiments, the amount of DNA methylation is ofclinical interest particularly in oncology. Aberrant DNA methylationpatterns (hypermethylation and hypomethylation compared to normaltissue) have been associated with a large number of human malignancies.Hypermethylation typically occurs at CpG islands in the promoter regionand is associated with gene inactivation. A lower level of leukocyte DNAmethylation is associated with many types of cancer (Zhang et al. (2011)Epigenetics, 6(3): 293-299). Global hypomethylation has also beenimplicated in the development and progression of cancer throughdifferent mechanisms. Typically, there is hypermethylation of tumorsuppressor genes and hypomethylation of oncogenes (see, e.g., Lund etal. (2004) J. Biol. Chem. 279(28): 29147-29154).

In this regard, it is noted that DNA methylation provides a prognosticindicator for Stage I Non-Small-Cell Lung Cancer (NSCLC). In particular,it was discovered that hypermethylation of five genes was significantlyassociated with shorter relapse-free survival (RFS) in stage I NSCLC:HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on thenumber of hypermethylated events distinguished patients with high- andlow-risk stage I NSCLC (see, e.g., Sandoval et al. (2013) J Clin.Oncol., 4140-4147).

Similarly it has been observed that malignant gliomas may have the MGMTgene inactivated due to methylation of its promoter region. Theprediction, born out by current research, is that by methylating theMGMT gene, a better response to chemotherapy can occur (as the tumor hasno means to repair the DNA damage induced by the alkylating agent). Ingliomas, MGMT promoter methylation is a favorable prognostic marker inthe setting of either radiation or chemotherapy (see, e.g.,//neurosurgery.ucsd.edu/brain-tumor-research-mgmt/).

By way of further illustration, Table 3 illustrates various genes thatare hypermethylated in certain cancers.

Table 3 shows illustrative, but non-limiting examples of geneshypermethylated in sporadic cancers (see, e.g., Baylin (2005) NatureClinical Practice Oncology, 2: S4-S11).

Gene or gene product Tumor type Rb Retinoblastoma APC Colorectal andother cancers p14/ARF Colorectal cancer p15/CDKN2B Leukemias p16/CDKN2AVarious cancers BRCA1 Breast, ovarian cancer VHL Renal cell cancershMLH1 Colorectal, gastric, endometrial cancers ER-α Breast, colorectal,other cancers

In various illustrative, but non-limiting, embodiments measurement ofmethylation of any one of more of the promoters of the following genesis contemplated: APC, ARF, CDKN2B, CDKN2A, BRCA1, VLH, hMLH1, MGMT.RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, AKR1B1,HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9.

Pancreatic Cancer.

In certain embodiments methylation status is determined for one or morepromoters where methylation status is a marker for the presence and/orprognosis of pancreatic cancer. It was determined that the frequency ofmethylation of one or more of ADAMTS1, or BNC1, can be used to detectand/or stage pancreatic cancer. Thus, illustrative, but non-limitingmethylation markers for pancreatic cancer include, but are not limitedto ADAMTS1 and/or BNC1. Illustrative primers and probes for thedetection of methylation at the promoters of these genes are shown inTable 4, below (referencing Table 5 for particular sequences), and inTable 12 in Example 4). In certain embodiments primers and probes areprovided for the detection of methylation in the forward strand of theconverted DNA and/or for the detection of methylation in the reversestrand of the converted DNA.

Breast Cancer.

In certain embodiments methylation status is determined for one or morepromoters where methylation status is a marker for the presence and/orprognosis of breast cancer. Illustrative methylation markers for breastcancer include, but are not limited to RASSF1A, and/or AKR1B1, and/orHOXB4, and/or HIST1H3C, and/or RASGRF2, and/or TM6SF1. Illustrativeprimers and probes for the detection of methylation at the promoters ofthese genes are shown in Table 4, below (referencing Table 5 forparticular sequences), and in Table 11 in Example 4.

In certain embodiments methylation status is determined for one or morepromoters where methylation status is a marker for the presence orlikelihood of lung cancer. Illustrative methylation markers for lungcancer include, but are not limited to CDO1, SOX17, TAC1, and/or HOXA7.

The methods described herein are not limited to determining methylationof the promoters of these genes. Using the methods described hereinmethylation of essentially any target of interest is possible.

It will be noted, however that measurement of DNA methylation need notbe limited to measurement of methylation at CPG islands in promoters.For example, it has been demonstrated that gene body methylation canalso alter gene expression and can provide a therapeutic target incancer (see, e.g., Yang et al. (2014) Cancer Cell, 26(4): 577-590).

Additionally, measurement of DNA methylation has prognostic/therapeuticapplications for pathologies other than cancer. For example, aberrantmethylation on regions on chromosomes 13, 18, 21, X, and Y can be usedto diagnose Down syndrome (see, e.g., Patsalis et al. (2012) Exp. Opin.Biol. Ther. 12(Suppl. 1): S155-S161). Because fetal DNA and maternal DNAare differentially methylated, cell-free DNA in maternal plasma canprovide a source of fetal DNA, which can be obtained non-invasively andutilized to assess the methylation state of the aforementionedchromosomes (or other chromosomes or genes).

As noted above, in certain embodiments, the cartridges and methodsdescribed herein are also used to determine mRNA levels, e.g., todetermine expression of various methyltransferases. In certainembodiments, expression level of RNA is determined for amethyltransferase selected from the group consisting of DNMT1, DNMT2,DNMT3A, DNMT3B, and TNMT3L.

Primers/Probes and Multiplex Analysis

In various embodiments the methods described herein can involve nestedPCR reactions and the cartridges described herein can contain reagents(e.g., primers and probes) for such nested PCR reactions. For example,in certain embodiments, methylation is detected for one, two, three,four, five, or six genes (gene promoters). Since bisulfite conversion ofa DNA changes cytosine resides to uracil, but leave 5-methyl cytosineresidues unaffected, the forward and reverse strands of converted(bisulfite-converted) DNA are no longer complementary. Accordingly, itis possible to interrogate the forward and reverse strands independently(e.g., in a multiplex PCR reaction) to provide additional specificityand sensitivity to methylation detection. In such instances, assaying ofa single target can involve a two-plex multiplex assay, while assayingof two, three, four, five, or six target genes can involve four-plex,six-plex, 8-plex, 10-plex, or 12-plex multiplex assays. In certainembodiments the assays can be divided into two multiplex reactions,e.g., to independently assay forward and reverse strands. However, itwill be recognized that when split into multiple multiplex assays, thegrouping of assays need not be by forward or reverse, but can simplyinclude primer/probe sets that are most compatible for particular PCRreaction conditions.

As indicated above, numerous cancers can be identified, and/or stagedand/or a prognosis therefor determined by the detection/characterizationof the methylation state on the forward and/or reverse strand of genepromoters whose methylation (or lack thereof) is associated with acancer. Illustrative gene (promoter) targets associated with variouscancers are described above and shown below in Table 4. It will berecognized that methylation (forward strand and/or reverse strand) ofone or more of the genes shown in Table 4 for each cancer can bedetermined to identify, and/or stage, and/or provide a prognosis for theindicated cancer. In certain embodiments methylation status of all ofthe genes shown for a particular cancer (forward and/or reverse strand)can be determined in a single multiplex PCR reaction.

Table 4. Illustrative primers and probes for the detection ofmethylation at the promoters of genes associated with various cancersusing the devices and methods described herein. Primer and probe numbersrefer to primer/probe numbers (primer/probe num) shown in Table 5,below.

External Internal Indication/Gene Primers Primers Probe Breast CancerAKR1B1:  58/183 19/20 193 HIST1H3C: 42/43, 59/54 194 HOXB4: 186/187,25/26 76 RASGRF2: 188/199, 192/14  67 RASSF1A: 189/1,  1/2 63 TM6SF1:202/51,  31/57 77 BG: 175/158, 176/156 164 Pancreatic Cancer Set 1: BNC1213/214, 221/222 229 ADAMTS1: 219/220, 227/228 265 ACTB: 102/103,320/321 150 Pancreatic Cancer Set 2: BNC1: 217/218, 225/226 264 ADAMTS1:215/216, 223/224 230 ACTB: 102/103, 320/321 150 Lung CPHD Set 2: CDO1:283/284, 287/288 291 TAC1: 293/294, 386/388 301 SOX17: 303/304, 382/385312 ACTB: 102/103, 320/321 150 Prostate: GSTP1: 233/234, 239/240 245APC: 235/236, 241/242 246 PTGS2: 237/238, 243/244 247 ACTB: 102/103,320/321 150 BRCA1: BRCA1: 328/329, 330/331 327 ACTB: 102/103, 320/321150 MGMT: MGMT: 248b/249b, 250/251 252 ACTB: 102/103, 320/321 150

Illustrative primers and probes for the detection of methylation at thepromoters of various genes are shown below in Table 5, below, and inTables 11 and 12 in Example 4. In certain embodiments these primersand/or probes are particularly suitable for use in a multiplexamplification.

TABLE 5 Illustrative primers and probes for the detection of methylation of various gene promoters. Primer/ Probe SEQNum Target Type Sequence ID NO   1 RASSF1A I GCGTTGAAGTCGGGGTTC 2   2RASSF1A I CCCGTACTTCGCTAACTTTAAACG 3   3 RASSF1A PACAAACGCGAACCGAACGAAACCA- 4 quencher/blocker   4 RASSF1A STD Ifluor-TTAGGGTAGATTGTGGATATTAG 5   5 RASSF1A STD IATACTAACAACTATCCAATACAAC 6   6 RASSF1A STD P fluor-(C*)AGGTTGAAATTAG(T-7 quencher)ATGTGTTATTTTGGTATGG   7 HIST1H3C I AATAGTTCGTAAGTTTATCGGCG 8  8 HIST1H3C I CTTCACGCCACCGATAACCGA 9   9 HIST4H3C P fluor- 10TACTTACGCGAAACTTTACCGCCGA- quencher/blocker  10 HIST1H3C STD IGATTTAGAGTTGGATGTGTGGAT 11  11 HIST1H3C STD I ACCACCATACTAATAATCAAATCTA12  12 HIST1H3C STD P fluor- 13 AAATATCACTCATCACCAAATAAATCCAA-quencher/blocker  13 RASGRF2 I GTAAGAAGACGGTCGAGGCG 14  14 RASGRF2 IACAACTCTACTCGCCCTCGAA 15  15 RASGRF2 P fluor- 16AACGAACCACTTCTCGTACCAACGA- quencher/blocker  16 RASGRF2 STD ITGTATGAGTTTGTGGTGAATAATG 17  17 RASGRF2 STD I AACTCACCATCAAACACTTTCCC 18 18 RASGRF2 STD P fluor- 19 TACAAACCCAACATCCTCTATCTATTC-quencher/blocker  19 AKR1B1 I GCGCGTTAATCGTAGGCGTTT 20  20 AKR1B1 ICCCAATACGATACGACCTTAAC 21  21 AKR1B1 P fluor- 22CGTACCTTTAAATAACCCGTAAAATCGA- quencher/blocker  22 AKR1B1 STD ITTTGTTGATGTTTTGTGGAAGTAAG 23  23 AKR1B1 STD I ATTCATCAATACTTTCAAATAACACA24  24 AKR1B1 STD P fluor-(C*)AAATACATTATCC(T- 25quencher)ACCACTAACAATACA  25 HOXB4 I CGGGATTTTGGGTTTTCGTCG 26  26 HOXB4I CGACGAATAACGACGCAAAAAC 27  27 HOXB4 P fluor- 28AACCGAACGATAACGAAAACGACGAA- quencher/blocker  28 HOXB4 STD IGTTAGTTTTGTAGTGTATTGAGTAT 29  29 HOXB4 STD I CATCTTCCACAATAAACTTCCAATT30  30 HOXB4 STD P fluor- 31 TAACTCCACCTATTCTACCTACCATTT-quencher/blocker  31 TM6SF1 I CGTTTAGCGGGATGCGGTGA 32  32 TM6SF1 IACACGAAAACCCCGATAACCG 33  33 TM6SF1 P fluor-AAACACTCATCGCAACCGCCGCG- 34quencher/blocker  34 TM6SF1 STD I TTAGATGTTGATTGGTTGTGTTTG 35  35TM6SF1 STD I ATCATCATAAAACTCAACAATCAATT 36  36 TM6SF1 STD P fluor- 37CCAAACATCAAATCTTTAACTTTTACCAA- quencher/blocker  37 RASSF1A STD Pfluor-AGGTTGAAATTAGTATG(T- 38 quencher)GTTATTTTGGTATGG- quencher/blocker 38 RASSF1A STD P fluor-AGGTTGAAATTAGTATGTGT(T- 39quencher)ATTTTGGTATGG-quencher/blocker  39 RASSF1A STD Pfluor-AGGTTGAAATTAGTATGTGTTA(T- 40 quencher)TTTGGTATGG-quencher/blocker 40 RASSF1A E GTTTTATAGTTTTTGTATTTAGG 41  41 RASSF1A EAACTCAATAAACTCAAACTCCC 42  42 HIST1H3C E GTGTGTGTTTTTATTGTAAATGG 43  43HIST1H3C E ATAAAATTTCTTCACRCCACC 44  44 RASGRF2 E GAGGGAGTTAGTTGGGTTAT45  45 RASGRF2 E CCTCCAAAAAATACATACCC 46  46 AKR1B1 EGTGTAATTAATTAGAAGGTTTTTT 47  47 AKR1B1 E AACACCTACCTTCCAAATAC 48  48HOXB4 E TTAGAGGYGAGAGAGTAGTT 49  49 HOXB4 E AAACTACTACTAACCRCCTC 50  50TM65F1 E AGGAGATATYGTTGAGGGGA 51  51 TM65F1 E TCACTCATACTAAACCRCCAA 52 52 RASSF1A STD I TTAGGGTAGATTGTGGATATTAGATAGG 53  53 RASSF1A STD ITAATACTAACAACTATCCAATACAACAC 54  54 HIST1H3C I CCGATAACCGAAACGCTCTTAC 55 55 AKR1B1 I GCGTTAATCGTAGGCGTTT 56  56 TM6SF1 I GTTTAGCGGGATGCGGTG 57 57 TM6SF1 I ACACGAAAACCCCGATAAC 58  58 AKR1B1 EGYGTAATTAAT(T*)AGAAGGITTTTT 59  59 HIST1H3C I TCGTACGAAGTAAATAGTTCGTAAG60  60 HIST1H3C E GGATTTTTGAAATATTATAGGATTAATTAG 61  61 RASSF1A EGTTTTATAGTT(T*)TTGTATTTAGG 62  62 RASSF1A P fluor- 63ACAAACGCGA(N*)ACCGAA(C**)GAAACCA- quencher/blocker  63 RASSF1A Pfluor-(C*)TGGTTTCGT(T- 64 quencher)CGGT(T*)CGCG-quencher/blocker  64RASSF1A STD P fluor-(C*)AGGTTGAAATTAGTA(T- 65quencher)GTGTTAT(T*)TTGG(T*)ATGG- quencher/blocker  65 HIST1H3C P fluor-66 CAAACTACTTACGCGAAACTT(T*)ACCG CC-quencher/blocker  66 HIST1H3C STD Pfluor- 67 AAATATCACTCA(T*)CACCAAA(N*)TAA A(T*)CCAA-quencher/blocker  67RASGRF2 P fluor- 68 AAACGAACCACTTCTCG(T*)ACCAACGAC- quencher/blocker  68RASGRF2 STD fluor- 69 P CAAACCCAACATCCTC(T*)ATC(T*)ATTC-quencher/blocker  69 AKR1B1 P fluor- 70 A(C*)GCGTACCTTT(N*)AAA(T*)AACCCG(T*)AAAATCG-quencher/blocker  70 AKR1B1 P fluor-A(C*)GCGTACCTT(T- 71quencher)AAA(T*)AACCCG(T*)AAAATCG- quencher/blocker  71 AKR1B1 STD Pfluor- (C*)AAATACATTATCC(T- 72 quencher)ACCAC(T*)AACAA(T*)ACA-quencher/blocker  72 HOXB4 P fluor- 73 AACCGAACGATAACGAAAA(C**)GACGA-quencher/blocker  73 HOXB4 STD P fluor- 74TAACTCCACCTATTC(T*)ACCT(N*)ACCA (T*)TT-quencher/blocker  74 TM6SF1 STD Pfluor- 75 CAAACATCAAATCT(T*)TAAC(T*)TT(T*) AC-quencher/blocker  75AKR1B1 P fluor- (C*)A(C*)GCGTACCT(T- 76quencher)TAAA(T*)AACCCG(T*)AAAATCG- quencher/blocker  76 HOXB4 P fluor-77 AACCGAACGA(T*)AACGAAA(N*)ACGACGAA- quencher/blocker  77 TM6SF1 Pfluor-AAACACTCATCGCAACCGCCGCG- 78 quencher/blocker  78 ACTB P fluor- 79TAACCACCACCCAACACA(C**)AATAAC- quencher/blocker  79 ALU Long Set 1 Pfluor- 80 CCCAACTACT(T*)AAAAAAC(T*)AAAAC- quencher/blocker  80ALU Short Set 1 P fluor- 81 CACCTAAAA(T*)CAAAAATT(T*)AAAACC-quencher/blocker  81 ALU Long Set 2 P fluor- 82CAAATAATTCTCC(T*)ACCTCAACC(T*)C- quencher/blocker  82 ALU Short Set 2 Pfluor- 83 CTTAACTCAC(T*)ACAACCTC(T*)ACC- quencher/blocker  83 INSL6 Pfluor- 84 CAAACCGAACGACGCGCACAAACAC- quencher/blocker  84 ACTB EGTATATAGGTTGGGGAAGTTTG 85  85 ACTB E AACTATACTCAACCAATAAAACC 86  86ALU Long Set 1 E TGTTATTTAGGTTGGAGTGTAG 87  87 ALU Long Set 1 ETAATAACTCATACCTATAATCCC 88  88 ALU Long Set 1 IGGTTGGAGTGTAGTGGTATAATTTTAG 89  89 ALU Long Set 1 ITAATAACTCATACCTATAATCCCAACAC 90  90 ALU Short Set 1 EGTAGAGATAGGGTTTTATTATGTTG 91  91 ALU Short Set 1 IGGTTTTATTATGTTGGTTAGGTTGG 92  92 ALU Long Set 2 E GTATTTTGGGAGGTTAAGGTAG93  93 ALU Long Set 2 E ATCTTACTCTTATTACCCAAAC 94  94 ALU Long Set 2 IGGTTAAGGTAGGTAGATTATTTGAGG 95  95 ALU Long Set 2 IATCTTACTCTTATTACCCAAACTAAAATAC 96  96 ALU Short Set 2 EGTTATTTAGGAGGTTGAGGTAG 97  97 ALU Short Set 2 EGAGGTAGGAGAATTATTTGAATTTAGG 98  98 INSL6 E ATTTGAGATTTTTGAGTTGG 99  99INSL6 E AACCCTACTCCCTATCTACG 100 100 INSL6 I GCGCGCGTTTTTTTTTGAAG 101101 INSL6 I GGCGTAGATAGGGAGTAGGGTT 102 102 ACTB IGTGATGGAGGAGGTTTAGTAAGTT 103 103 ACTB I CCAATAAAACCTACTCCTCCCTTAA 104104 RASSF1A STD P fluor-(C*)(C*)ATACCAAAA(T- 105quencher)AACACA(T*)CTAAT(T*)TCAACCT- quencher/blocker 105 AKR1B1 STD Pfluor-(C*)AAATACAT(T*)ATCC(T- 106 quencher)ACCAC(T*)AACAA(T*)ACA-quencher/blocker 106 AKR1B1 P fluor-(C*)ACGCGTACCTT(T- 107quencher)AAA(T*)AACCCG(T*)AAAATCG- quencher/blocker 107 AKR1B1 Pfluor-(C*)ACGCGTACCTT(T*)AAA(T- 108 quencher)AACCCG(T*)AAAATCG-quencher/blocker 108 RASSF1A UM P fluor-CTAACAAACA(C- 109quencher)AAA(C**)CAAA(C**)AAAACCA- quencher/blocker 109 RASSF1A UM Pfluor-CTAACAAACA(C**)AAA(C- 110 quencher)CAAA(C**)AAAACCA-quencher/blocker 110 HIST1H3C UM P fluor- 111AACTACTTACA(C**)AAAACTT(N*)TAC(C**) ACCAA-quencher/blocker 111HIST1H3C UM P fluor- 112 AACTACTTA(C**)ACAAAA(C**)TTTACCAC-quencher/blocker 112 RASGRF2 UM P fluor- 113AAACAAACCAC(T*)TCTCA(T*)ACCAACAAC- quencher/blocker 113 AKR1B1 UM Pfluor-(C*)ACATACCTTTAAA(T- 114 quencher)AACCCA(T*)AAAA(T*)CAAC-quencher/blocker 114 AKR1B1 UM P fluor-(C*)ACATACCTT(T- 115quencher)AAA(T*)AACCCA(T*)AAAATCA AC-quencher/blocker 115 HOXB4 UM Pfluor- 116 CAACAAAAACCCAAAA(T*)CCCAAC(N*) AAACCACA-quencher/blocker 116HOXB4 UM P fluor- 117 CAAAATCCCAA(C**)AAACCA(C**)ATAACA-quencher/blocker 117 TM6SF1 UM P fluor- 118AAACACTCATCACAACCA(C**)CACACC- quencher/blocker 118 AKR1B1 UM ITGGTGTGTTAATTGTAGGTGTTTT 119 119 AKR1B1 UM I CCCAATACAATACAACCTTAACC 120120 HOXB4 UM I GTGGTGTGTATTGTGTAGTGTTA 121 121 HOXB4 UM ICAAACCAAACAATAACAAAAACAAC 122 122 TM6SF1 UM I TGTTTAGTGGGATGTGGTGAAG 123123 TM6SF1 UM I ACACAAAAACCCCAATAACCACA 124 124 RASSF1A UM IGTTTAAAGTTAGTGAAGTATGGGTTT 125 125 HIST1H3C UM ITGTATGAAGTAAATAGTTTGTAAGTTTATTGG 126 126 AKR1B1 STD ITTTGTTGATGTTTTGTGGAAG(T*)AAG 127 127 AKR1B1 STD IATTCATCAATACTTTCAAA(T*)AACACA 128 128 RASGRF2 P fluor- 129AAACGAACCACTTCTCG(T*)ACCAACGAC- quencher/blocker 129 RASGRF2 STD Pfluor- 130 CAAACCCAACATCCTC(T*)ATC(T*)ATTC- quencher/blocker 130 TM6SF1P fluor-AAACACTCATCGCAACCGCCGCG- 131 quencher/blocker 131 TM6SF1 STD Pfluor- 132 CCAAACATCAAATCT(T*)TAACTT(T*)TA CCAA-quencher/blocker 132TM6SF1 P fluor-AAACACTCATCGCAACCGCCGCG- 133 quencher/blocker 133RASSF1A UM I GGTGTTGAAGTTGGGGTTTG 134 134 RASSF1A UM ICCCATACTTCACTAACTTTAAAC 135 135 HIST1H3C UM I GTAAATAGTTTGTAAGTTTATTGGTG136 136 HIST1H3C UM I TTTCTTCACACCACCAATAACCAA 137 137 RASGRF2 UM IGAGTAAGAAGATGGTTGAGGTG 138 138 RASGRF2 UM I CAACAACTCTACTCACCCTCAA 139139 P fluor- 140 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC- quencher/blocker 140ALU Long Set 1 P fluor- 141 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC-quencher/blocker 141 ALU Long Set 1 P fluor- 142TCCCAACTACT(T*)AAAAAAC(T*)AAAAC- quencher/blocker 142 ALU Long Set 1 Pfluor- 143 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC- quencher/blocker 143ALU Long Set 1 P fluor- 144 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC-quencher/blocker 144 HMBS I GGATAAGATTTTTGATATTGTATTTTTTAAGG 145 145HMBS I CATATTCAAACTCCTTAATAAACAAACTTTTCTC 146 146 HMBS Pfluor-CCGAACAAAAAAAA(C- 147 quencher)CTAAA(T*)AAATCCC(T*)TC-quencher/blocker 147 ACTB P fluor- 148 CCACCACCCAACACACAA(T*)AACAAACAC-quencher/blocker 148 ACTB I GGTTTAGTAAGTTTTTTGGATTGTG 149 149 ACTBCCTTAAAAATTACAAAAACCACAAC 150 150 ACTB P fluor-CCACCACCCAACA(C- 151quencher)ACAA(T*)AACAAACAC- quencher/blocker 151 ACTB P fluor- 152CCACCACCCAAC(N*)ACA(C**)AATAA(C**) AAACAC-quencher/blocker 152 ACTB Pfluor- 153 CCACCACCCAACACA(N*)CAA(T*)AACAAACAC- quencher/blocker 153 BGI TTCAGTGCCGGTTGGTAATGTAA- 154 quencher/blocker 154 BG ICAACAACTTTAATACCTGTTTCAAGGA 155 155 BG conv I GGTATTTTTGTATTTGTTGGTGTTG156 156 BG conv I CATACATACACCAAACAATTCATTC 157 157 BG conv EGTATGGTGGTATTTTTGTATTTGTTG 158 158 BG conv E CACACATACATACACCAAACAATTC159 159 BG P fluor- 160 AAGATCCGATTCACAGA(N*)CAAGCTCCGTCA-quencher/blocker 160 BG I fluor- 161 AAGATCCGATTCACAGA(N*)CAAGCTCCGTCA-quencher/blocker 161 BG conv P fluor-(C*)AAAATCATTT(C- 162quencher)CTT(C**)ACAAATA(C**)ACTC- quencher/blocker 162 BG conv Pfluor-CCAAATACCA(T- 163 quencher)AACCA(T*)TTTATTAA(T*)AACAC-quencher/blocker 163 BG conv P fluor- 164AAAATCATTTCCTT(C**)ACA(N*)AATA(C**) ACTC-quencher/blocker 164 BG conv Pfluor- 165 CCAAATACCA(T*)AACCAT(N*)TTTATTA A(T*)AACAC-quencher/blocker165 short HMBS I CCCTAGTATGCTAGGTCTCTTGCTGGGA 166 166 short HMBS ICAGCCTCTCTGAGGGTTTAAGCCCA 167 167 short HMBS P fluor- 168TCAGCC(T*)ATC(T*)GACACCCCGGG- quencher/blocker 168 short β-Globin IGACTCCTGAGGAGAAGTCTGCCGTTA 169 169 short β-Globin ICCTTGATACCAACCTGCCCAGGG 170 170 short β-Globin P fluor- 171AGGTGAACG(T*)GGATGAAGT(T*)GGTGGTG- quencher/blocker 171 short BG ICAACATCGCGCAAGAGCACGG 172 172 short BG I CGTTTCCTTCACGAGTACGCTCTCCGA 173173 short BG P fluor- 174 ACCGGCGAA(T*)ACAGAGA(T*)ACCG- quencher/blocker174 ACTB P fluor- 175 CC(A*)CC(A*)CCCAAC(N*)ACA(C**)AATAA(C**)AAACAC-quencher/blocker 175 BG conv I GTTGGTGTTGGAGAGTGTATTTG 176176 BG conv I GGAGAGTGTATTTGTGAAGGAAATG 177 177 BG conv IGGAAATGATTTTTTTTATGAGATGAGTG 178 178 ACTB P fluor- 179CCACCACCCAACACA(N*)CAA(T*)AACAAACAC- quencher/blocker 179 ACTB P fluor-180 CCACCACCCAACACA(N*)CAA(T*)AACAAACAC- quencher/blocker 180 ACTB Pfluor- 181 CCACCACCCAACACACAA(T*)AACAAACAC- quencher/blocker 181 ACTB IGATGGAGGAGGTTTAGTAAGTTTTT 182 182 ACTB I AATAAAACCTACTCCTCCCTTAAAAA 183 183a AKR1B1 E CTTACCATAACTACTAC(dK)CTCC 184  183b AKR1B1 ECTTACCATAACTACTACRCTCC 185 184 HIST1H3C E GTGTGTGTTTTTATTGTAAATGGT 186 185a HIST1H3C E AAC(dK)ATAAC(dK)ATAAAATTTCTTCAC 187  185b HIST1H3C EAACRATAACRATAAAATTTCTTCAC 188  186a HOXB4 E GTTTGT(dP)GGGATTTTGGGT 189 186b HOXB4 E GTTTGTYGGGATTTTGGGT 190  187a HOXB4 ECC(dK)AACTCC(dK)AAAAAAAAACC 191  187b HOXB4 E CCRAACTCCRAAAAAAAAACC 192 188a RASGRF2 E GGTATTAAG(dP)G(dP)GGTTTTTTG 193  188b RASGRF2 EGGTATTAAGYGYGGTTTTTTG 194  189a RASSF1A E GT(dP)GTTTAGTTTGGATTTTGG 195 189b RASSF1A E GTYGTTTAGTTTGGATTTTGG 196 190 TM6SF1 ETTTCGAAGGGTAAGCGTTAAG 197  191a TM6SF1 E AACATAAATAACC(dK)AAA(T*)AACC198  191b TM6SF1 E AACATAAATAACCRAAA(T*)AACC 199 192 RASGRF2 ICGGTTTTTTGAGTAAGAAGACGGTC 200  193a AKR1B1 P fluor-TACCTTTAAA(T- 201quencher)AACCC(dK)(T*)AAAA(T*)CGACAA- quencher/blocker  193b AKR1B1 Pfluor-TACCTTTAAA(T- 202 quencher)AACCCR(T*)AAAA(T*)CGACAA-quencher/blocker  194a HIST1H3C P fluor- 203ATAACAAACTACT(T*)AC(dK)CGAAAC(T*)TTAC- quencher/blocker  194b HIST Pfluor- 204 ATAACAAACTACT(T*)ACRCGAAAC(T*)TTAC- quencher/blocker  195aHOXB4 P fluor- 205 AACAAACC(dK)AA(C**)GA(T*)AAC(N*)AAAAC-quencher/blocker  195b HOXB4 P fluor- 206AACAAACCRAA(C**)GA(T*)AAC(N*)AAAAC- quencher/blocker 196 RASGRF2 Pfluor- 207 CACATTCTAA(T*)AAAAAAC(N*)AACCAC(T*)TC- quencher/blocker  197aRASSF1A P fluor-AACC(dK)AA(C**)GAAA(C- 208quencher)CA(C**)AAAAC-quencher/blocker  197b RASSF1A Pfluor-AACCRAA(C**)GAAA(C- 209 quencher)CA(C**)AAAAC-quencher/blocker 198TM6SF1 P fluor- 210 CAAAAACAC(T*)CATC(N*)CAACCGCC- quencher/blocker 199RASGRF2 E ACAACCCTCCAAAAAATACATA 211 200 BG conv P fluor- 212CCAAATACCATAACCA(T*)TTTATTAA(T*)AACAC- quencher/blocker 201 BG conv Pfluor- 213 CCAAATACCATAACCA(T*)TTTATTAA(T*)AACAC- quencher/blocker  202aTM6SF1 E TTT(dP)GAAGGGTAAG(dP)GTTAAG 214  202b TM6SF1 ETTTYGAAGGGTAAGYGTTAAG 215  203a TM6SF1 E CAACAC(dK)AAAACCCC(dK)ATA 216 203b TM6SF1 E CAACACRAAAACCCCRATA 217 204 KRAS Multi ECCTGCTGAAAATGACTGAATATAACCGC 218 TAAGAACCTCTCGGTCAGCTGAT 205 KRAS MultiE CCTGCTGAAAATGACTGAATATAAAGTC 219 TCATTATAATCGTTCGAGCTGTT 206KRAS Multi E CCTGCTGAAAATGACTGAATATAAGCAG 220 ACTTGGCGGTAGGTCCGAGCTTG207 KRAS Multi E CCTGCTGAAAATGACTGAATATAAGTAT 221CCTGAGCACGGTTGCGAGCTGCT 208 KRAS Multi I CTCTTGCCTACGCC(N*)CCGCTAAGAACC222 TCTCGGTC 209 KRAS Multi I CTCTTGCCTACGCC(N*)AGTCTCATTATA 223ATCGTTCG 210 KRAS Multi I CTCTTGCCTACGCC(N*)GCAGACTTGGCG 224 GTAGGTCC211 KRAS Multi I CTCTTGCCTACGCC(N*)GTATCCTGAGCA 225 CGGTTGCG 212 ACTB Pfluor- 226 CCACCACCCAACACACAA(T*)AACAAACAC- quencher/blocker 213 BNC1 ECCCRCAAACCRCGAAAACCTC 227 214 BNC1 E GTTTTTTTTYGGGAGAGGTAAATA 228 215ADAMTS1 E CRCCTCCRAAACTAAAACAAC 229 216 ADAMTS1 E GGGTTATTGTAAAGTTAGGGTG230 217 BNC1 E GAGGT(dP)GTGGTTTT(dP)GTAGAT 231 218 BNC1 EAAAC(dK)CCAAAAAACTTCAAAAC 232 219 ADAMTS1 E TTTTGTTGGGATAAGAAG(dP)GTTT233 220 ADAMTS1 E ACCAAAAACTATTACAAAACCAAA 234 221 BNC1 I CCGACGACCGACG235 222 BNC1 I GGGAGAGGTAAATATCGATAC 236 223 ADAMTS1 ICGCGAAAATTAATACCTAACG 237 224 ADAMTS1 I TTAGGGTGCGTTATCGGAC 238 225 BNC1I CGGAGGTGTTTGTTTTCGTC 239 226 BNC1 I CGAAAAAAACAAACACCGACACG 240 227ADAMTS1 I CGTTTTCGGGGTTGAGGTAAC 241 228 ADAMTS1 I CCAAAATACGCTACCGAACGA242 229 BNC1 P fluor- 243 AAAAT(A*)TCT(A*)(C**)CCC(C**)(dK)CC-quencher/blocker 230 ADAMTS1 P fluor- 244TATTACTCACTCTAC(T*)CAAAAC(T*)CTCC- quencher/blocker 231 BNC1 P fluor-245 ATATCTTTTACCAACAAA(T*)ACCT(T*)CAAA- quencher/blocker 232 ADAMTS1 Pfluor- 246 GTTTT(dP)GTTTTGGTTGCGA(T*)GTTGT- quencher/blocker 233 GSTP1 EGGGATTTTTTAGAAGAG(dP)GGT 247 234 GSTP1 E TACTCACTAATAAC(dK)AAAAC(T*)AC248 235 APC E GGTTTTGTGTTTTATTG(dP)GGAG 249 236 APC ECCTAAC(dK)AACTACACCAATACAA 250 237 PTGS2 E GAGAGGGGATTTTTTG(dP)GTTT 251238 PTGS2 E CC(dK)AAAACCAATTCTAAACTAATC 252 239 GSTP1 ITTTTTAGAAGAGCGGTCGGC 253 240 GSTP1 I CTAATAACGAAAACTACGACGACG 254 241APC I TTGTGTTTTATTGCGGAGTGC 255 242 APC I AACCACATATCGATCACGTACG 256 243PTGS2 I GCGTTTTCGGATTTTAGGGTC 257 244 PTGS2 I AACTAATCGCCTTAAATAAAATACCG258 245 GSTP1 P fluor- 259 CCTCC(dK)AACCTTA(T*)AA(N*)AAA(T*)AATCCC-quencher/blocker 246 APC P fluor- 260AAAAAC(dK)CCCTAATCC(N*)CA(T*)CCAAC- quencher/blocker 247 PTGS2 P fluor-261 CACTTAACTTCCTC(T*)CCAAAAATC(T*)AAAC- quencher/blocker  248a MGMT EGTTTT(T*)AGAA(dP)G(T*)TTTG(dP)GTTT 262  248b MGMT EGTTTT(T*)AGAAYG(T*)TTTGYGTTT 263  249a MGMT E AAAAAAC(T*)CC(dK)CACTCTTCC264  249b MGMT E AAAAAAC(T*)CCRCACTCTTCC 265 250 MGMT ITTTCGACGTTCGTAGGTTTTCGC 266 251 MGMT I GCACTCTTCCGAAAACGAAACG 267  252aMGMT P fluor- 268 CCAAACAC(T*)CACCAAATC(N*)CAAAC- quencher/blocker  252bMGMT P fluor- 269 CCAAACAC(T*)CACCAAATC(N*)CAAAC- quencher/blocker 264BNC1 P fluor-ATATCTTTTACCAA(C- 270 quencher)AAA(T*)ACCT(T*)CAAA-quencher/blocker 265 ADAMTS1 P fluor- 271GTTTT(dP)GTTTTGGTTGCGA(T*)GTTGT- quencher/blocker 283 CDO1 EGGAGATAA(dP)GGGGTTTTTGG 272 284 CDO1 E CACTAAAAATATACCAAC(dK)ACC 273 285CDO1 E GGAGAGTTATTTAAGAAAGGTGG 274 286 CDO1 E AAAATTAC(dK)C(dK)AAACCCAC275 287 CDO1 I CGTGTTCGTAGGGTTTTTTCGTTTTC 276 288 CDO1 ICCAACGACCCTCGAAAAAAAAACG 277 289 CDO1 I GATTTTGCGGGTACGGTTTACGC 278 290CDO1 I GATCCCTAAAACGCCGAAAACAACG 279 291 CDO1 P fluor-(C*)GTTATTTTT(T-280 quencher)TTGGG(T*)GGTT(T*)TTCG- quencher/blocker 292 CDO1 Pfluor-C(dK)AAAAACCACC(C- 281 quencher)AAAAAAAA(T*)AAC- quencher/blocker293 TAC1 E GGATAAATAT(dP)GTAAGGTATTGAG 282 294 TAC1 ECGAAATACTAAATTTCTCTAATTCCTC 283 295 TAC1 E GAGTTTTTTTGGTTTTTT(dP)GAG 284296 TAC1 E CTAAAATAAATACC(dK)CAAAACAC 285 297 TAC1 ICGCGTTCGGATTTTTTTTTCGGC 286 298 TAC1 I AAATTTCTCTAATTCCTCCGAACGCACG 287299 TAC1 I GCGTACGTTGGTCGTTTCGTATTTTC 288 300 TAC1 IGCAAAACACTAAACAAACGAAAAAACGCG 289 301 TAC1 P fluor- 290GTAGTTAT(dP)GAGAG(T*)G(N*)GGAGCG A(T*)TAG-quencher/blocker 302 TAC1 Pfluor- 291 CTAATC(dK)CTCCGCAC(T*)CTC(N*)A(T*) AACTAC-quencher/blocker303 SOX17 E GTTTGGAG(dP)GTTATGAGTAG 292 304 SOX17 ECTTCATATCCCC(dK)ATAAAACTC 293 305 SOX17 E GGGTTTTTAGTCGGTTTAGTG 294 306SOX17 E CTAAAAC(dK)TAAAACTC(dK)AACC 295 307 SOX17 IGATTTAGAGCGCGTTGTTCGC 296 308 SOX17 I CATATCCCCGATAAAACTCAACGACTCG 297309 SOX17 I GTCGGTTTAGTGATATTGCGGGC 298 310 SOX17 ICCACGACCTAAACGTAAACCTAACG 299 311 SOX17 P fluor- 300GATGGT(dP)GGGTTGGGTT(T*)TTGTTTTTGG- quencher/blocker 312 SOX17 P fluor-301 CCAAAAACAAAAACCCAA(C**)CCGACCATC- quencher/blocker 313 CD01 P fluor-302 (C*)GTATATTTT(dP)GGTT(T*)TTT(N*)GG GT(T*)TCG-quencher/blocker 314CD01 P fluor- 303 C(dK)AAACC(C**)GAAAAAA(C**)C(N*)AAAATATAC-quencher/blocker 315 TAC1 P fluor- 304GGTAGTTGT(dP)G(T*)CGGGAAGGAGGTTCG- quencher/blocker 316 TAC1 P fluor-305 C(dK)AACCTCCTTCCCGAC(N*)ACAAC(T*)ACC- quencher/blocker 317 SOX17 Pfluor- 306 GGTTTTTTTTGTA(T*)AGATGTGGT(T*)AATGG- quencher/blocker 318SOX17 P fluor- 307 CCATTAACCACA(T*)CTA(T*)ACAAAAAAAACC- quencher/blocker319 SOX17 E GGTTTGGTTTATAG(dP)GTATTTAGG 308 320 ACTB IGAGGTTTAG(T*)AAGTTTTTTGGATTGTG 309 321 ACTB ICCCTTAAAAAT(T*)ACAAAAACCACAAC 310 322 BRCA1 E GTAGATTGGGTGGTTAATTTAGAG311 323 BRCA1 E CTATAATTCCC(dK)C(dK)CTTTTC 312 324 BRCA1 IGGTGGTTAATTTAGAGTTTCGAGAGAC 313 325 BRCA1 I CGTTACCACGAAAACCAAAAAACTACCG314 326 BRCA1 P fluor- 315 GATTTCGTATTT(T*)GAGAGG(T*)TGTTGTTTAG-quencher/blocker 327 BRCA1 P fluor- 316CTAAACAACAACC(T*)CTCAAAA(T*)AC GAAATC-quencher/blocker 328 BRCA1 EGGTAGATTGGGTGGTTAATTTAGAG 317 329 BRCA1 E CCAAAAAATCTCAACRAACTC 318 330BRCA1 I GGGTGGTTAATTTAGAGTTTCGAGAGAC 319 331 BRCA1 IACCACGAAAACCAAAAAACTACCG 320 336 MGMT E GGGATTTTGTTTAAGTATGTTAAAGG 321337 MGMT E CCTACCTTACCTCTAAATACCAACC 322 338 MGMT IGTATGTTAAAGGGTTGTTGTAAGTTAAGG 323 339 MGMT I CCTCTAAATACCAACCCCAAACC 324340 MGMT P fluor-CCAACTACTC(C- 325 quencher)AAAAAACTTCCAAAAACC-quencher/blocker 341 MGMT P fluor-CCAAC(T*)ACTC(C- 326quencher)AAAAAAC(T*)TCCAAAAACC- quencher/blocker 342 MGMT IGTATGTTAAAGGGTTGT(T*)GTAAGTTAAGG 327 343 MGMT ICCTCTAAATACCAA(C**)CCCAAACC 328 380 ACTB P fluor- 329CCACCACCCAACACACAA(T*)AACAAACAC- quencher/blocker 381 ACTB P fluor- 330CCACCACCCAACACACAA(T*)AACAAACAC- quencher/blocker 382 SOX17 IATTTAGAGCGCGTTGTTCGC 331 383 SOX17 I ATATCCCCGATAAAACTCAACGACTCG 332 384SOX17 I TATCCCCGATAAAACTCAACGACTCG 333 385 SOX17 IATCCCCGATAAAACTCAACGACTCG 334 386 TAC1 I GCGTTCGGATTTTTTTTTCGGC 335 387TAC1 I TTTCTCTAATTCCTCCGAACGCACG 336 388 TAC1 I CTCTAATTCCTCCGAACGCACG337 389 SOX17 I GTGACGATTAGAGTTAGATTTAGAGCGC 338 390 TAC1 P fluor- 339GTAGTTATCGAGAG(T*)GCGGAGCGA(T*)TAG- quencher/blocker 391 SOX17 P fluor-340 CCAACCCGACCATCACCGCGAACAAC- quencher/blocker 392 BG converted IGGAGAGTGTATTTG(T*)GAAGGAAATG 341 393 BG converted ICATACATACACCAAACAA(T*)TCATTC 342 394 BG converted P fluor- 343CCAAATACCA(T*)AACCATTTTATTAA(T*) AACAC-quencher/blocker 395 BG convertedP fluor- 344 CCAAATACCA(T*)AACCATTTTATTAA(T*) AACAC-quencher/blocker 396GSTP1 (Fwd) E GGTTTYGTTGGGGATTTG 345 397 GSTP1 (Fwd) EACCRCTCTTCTAAAAAATCC 346 398 GSTP1 (Fwd) I AGGTTTTTTCGGTTAGTTGCGC 347399 GSTP1 (Fwd) I AACGTCGACCGCAAAAAAACG 348 400 GSTP1 (Fwd) Pfluor-(C*)GCGAT(T*)T(C- quencher)GGGGA(T*)T(T*)TAGG- 349quencher/blocker 401 GSTP1 (Fwd) P fluor-CC(T*)AAAA(T*)C(C- 350quencher)CCGAAA(T*)CGC-quencher/blocker 402 APC (Fwd) EGAAGTAGTTGTGTAATTYGTTGG 351 403 APC (Fwd) E CACCCTAACRAACTACACC 352 404APC (Fwd) I TGCGGATTAGGGCGTTTTTTATTTTC 353 405 APC (Fwd) ITACAACCACATATCGATCACGTACG 354 406 APC (Fwd) Pfluor-GGAGTTCGTCGA(T*)TGG(T*)TGGG- 355 quencher/blocker 407 APC (Fwd) Pfluor-CCCAACCAA(T*)CGACGAAC(T*)CC- 356 quencher/blocker 408 EYA4 (Fwd) EGAGTTTTTYGGAGGGTTATAG 357 409 EYA4 (Fwd) E CAAACTACAAAAAACATTCAATCC 358410 EYA4 (Fwd) I GCGTTTGGGTTTTTTCGGTGTC 359 411 EYA4 (Fwd) IATCGCCGCAATTAAAAAACCCG 360 412 EYA4 (Fwd) P fluor- 361GGTTCGCGTTTTAAT(T*)TTTAGG(T*)ATTG- quencher/blocker 413 EYA4 (Fwd) Pfluor- 362 CAATACC(T*)AAAAAT(T*)AAAACGCGAACC- quencher/blocker 414OLIG2(Fwd) E GTTATGGATTYGGAYGTTAG 363 415 OLIG2(Fwd) ECTCCRACRAACAATCACTC 364 416 OLIG2(Fwd) I GTTTGGTGTTTAG(T*)CGTTCGTC 365417 OLIG2(Fwd) I CACTCGAAATAAA(C**)GAAAACACG 366 418 OLIG2(Fwd) P fluor-367 GGTAGTAGCGG(T*)AGCGTT(T*)TTATTG- quencher/blocker 419 OLIG2(Fwd) Pfluor- 368 CAATAAAAACGC(T*)ACCGC(T*)ACTACC- quencher/blocker 420ADAMTS12(Fwd) E GGYGTAGTTTATTTYGGTT 369 421 ADAMTS12(Fwd) EATTTAACCRACTCRACCAAC 370 422 ADAMTS12(Fwd) I GTATGTTTCGCGGTTTCGTAGTTC371 423 ADAMTS12(Fwd) I ACTAAACCTAACG(T*)TCGAAACG 372 424 ADAMTS12(Fwd)P fluor-(C*)GTTCGTTCGG(T- 373 quencher)G(T*)ATTTTTT(T*)TTCGG-quencher/blocker 425 ADAMTS12(Fwd) P fluor-CCGAAAAAAAAA(T- 374quencher)A(C**)ACCGAA(C**)GAAC- quencher/blocker 426 POU4F1(Fwd) EGTTTGAGTTGTTTTGATTTTAGTG 375 427 POU4F1(Fwd) E CTCCAACCTCAACTCTAAAC 376428 POU4F1(Fwd) I GATTTTAGTGTCGCGTATTTTGGTTC 377 429 POU4F1(Fwd) ICTAAACTAAATCCCGCGAACCTCG 378 430 POU4F1(Fwd) P fluor- 379GGTTTTAT(T*)GGGGGTT(N*)AT(T*)TCG GGTAG-quencher/blocker 431 POU4F1(Fwd)P fluor- 380 CTACCCGAAATAACCC(C**)CAA(N*)TAA AA(C**)C-quencher/blocker432 ABCB1(Fwd) E GGTTTTTAGTATTTTTAYGAAGGT 381 433 ABCB1(Fwd) ECRATACRAAAACCTACTCTCTA 382 434 ABCB1(Fwd) I TTTGGATTTTGTTCGTCGTTAGTGC383 435 ABCB1(Fwd) I CTACTCTCTAAACCCGCGAACG 384 436 ABCB1(Fwd) P fluor-385 GGTTTTAGTCG(T*)CGCGGACGATGT- quencher/blocker 437 ABCB1(Fwd) Pfluor- 386 ACATCGTCCGCGACGAC(T*)AAAACC- quencher/blocker 438 SOX17 IGAGTTAGATTTAGAGCGCGTTGTTC 387 439 TAC1 I GAGCGCGTTCGGATTTTTTTTTC 388Note Y is C/T; R is A/G; C* is an optionally functionalized (e.g., toalter probe T_(m)) C; T* is an optionally functionalized (e.g., to alterprobe T_(m)) T; A* is an optionally functionalized (e.g., to alter probeT_(m)) A; N* is a nucleotide optionally a quencher; dP is a universalpyrimidine; dK is a universal purine.

It is noted that these primers and probes identify the locations ofvarious fluorophores and quenchers. However, it will be recognized thatthe particular fluorophores and quenchers are illustrative and notlimiting and numerous amplification and/or detection strategies can beemployed in the cartridges described herein. Accordingly, in variousembodiments the methods and devices described herein can employ manydifferent nucleic acid hybridization probes. Typically, for signalgeneration, the probes utilize a change in the fluorescence of afluorophore due to a change in its interaction with another molecule ormoiety brought about by changing the distance between the fluorophoreand the interacting molecule or moiety. Alternatively, other methods ofdetecting a polynucleotide in a sample, including, but not limited to,the use of radioactively-labeled probes, are contemplated.

Fluorescence-based assays typically rely for signal generation onfluorescence resonance energy transfer, or “FRET”, according to which achange in fluorescence is caused by a change in the distance separatinga first fluorophore from an interacting resonance energy acceptor,either another fluorophore or a quencher. Combinations of a fluorophoreand an interacting molecule or moiety, including quenching molecules ormoieties, are known as “FRET pairs.” The mechanism of FRET-pairinteraction typically requires that the absorption spectrum of onemember of the pair overlaps the emission spectrum of the other member,the first fluorophore. If the interacting molecule or moiety is aquencher, its absorption spectrum typically overlaps the emissionspectrum of the fluorophore (see, e.g., Stryer (1978) Ann. Rev. Biochem.47: 819-846; Selvin (1995) Meth. Enzymol. 246: 300-335; and the like).Efficient FRET interaction is typically achieved when the absorption andemission spectra of the pair have a large degree of overlap. Theefficiency of FRET interaction is linearly proportional to that overlap.Typically, a large magnitude of signal (i.e., a high degree of overlap)is desired. FRET pairs, including fluorophore-quencher pairs, aretherefore typically chosen on that basis.

A variety of labeled nucleic acid hybridization probes and detectionassays that utilize FRET and FRET pairs are known. One such scheme isdescribed by Cardullo et al. (1988) Proc. Natl. Acad. Sci. USA, 85:8790-8794 and in Heller et al. EP 0070685. It uses a probe comprising apair of oligodeoxynucleotides complementary to contiguous regions of atarget DNA strand. One probe molecule contains a fluorescent label, afluorophore, on its 5′ end, and the other probe molecule contains adifferent fluorescent label, also a fluorophore, on its 3′ end. When theprobe is hybridized to the target sequence, the two labels are broughtvery close to each other. When the sample is stimulated by light of anappropriate frequency, fluorescence resonance energy transfer from onelabel to the other occurs. FRET produces a measurable change in spectralresponse from the labels, signaling the presence of targets. One labelcould be a “quencher,” which can be, inter alia, an interactive moiety(or molecule) that releases the accepted energy as heat.

Another type of nucleic acid hybridization probe assay utilizing a FRETpair is the “TaqMan®” assay described in Gelfand et al. U.S. Pat. No.5,210,015, and Livak et al. U.S. Pat. No. 5,538,848. The probe istypically a single-stranded oligonucleotide labeled with a FRET pair. Ina TaqMan® assay, a DNA polymerase releases single or multiplenucleotides by cleavage of the oligonucleotide probe when it ishybridized to a target strand. That release provides a way to separatethe quencher label and the fluorophore label of the FRET pair.

In certain embodiments non-FRET fluorescent probes, such as thosedescribed in, e.g., Tyagi et al., U.S. Pat. No. 6,150,097 can also beused. For example, the Tiyagi et al. patent describes how changes in theabsorption spectra of the label pair can be used as a detectable signalas an alternative to change in fluorescence. When change in absorptionis utilized, the label pair may include any two chromophores, that is,fluorophores, quenchers and other chromophores. The label pair may evenbe identical chromophores.

In some embodiments, dyes and other moieties, such as quenchers, areintroduced into primers and/or probes used in the methods and cartridgesdescribed herein. In certain embodiments such dyes and quenchersinclude, but are not limited to dyes (fluors) suitable for use as FRETprobes. In certain embodiments the dyes and/or quenchers comprisemodified nucleotides. A “modified nucleotide” refers to a nucleotidethat has been chemically modified, but still functions as a nucleotide.In some embodiments, the modified nucleotide has a chemical moiety, suchas a dye or quencher, covalently attached, and can be introduced into apolynucleotide, for example, by way of solid phase synthesis of thepolynucleotide. In some embodiments, the modified nucleotide includesone or more reactive groups that can react with a dye or quencherbefore, during, or after incorporation of the modified nucleotide intothe nucleic acid. In some embodiments, the modified nucleotide is anamine-modified nucleotide, i.e., a nucleotide that has been modified tohave a reactive amine group. In some embodiments, the modifiednucleotide comprises a modified base moiety, such as uridine, adenosine,guanosine, and/or cytosine. In some embodiments, the amine-modifiednucleotide is selected from 5-(3-aminoallyl)-UTP;8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP;N6-(4-amino)butyl-ATP, N6-(6-amino)butyl-ATP,N4-[2,2-oxy-bis-(ethylamine)]-CTP; N6-(6-Amino)hexyl-ATP;8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP,5-propargylamino-UTP. In some embodiments, nucleotides with differentnucleobase moieties are similarly modified, for example,5-(3-aminoallyl)-GTP instead of 5-(3-aminoallyl)-UTP. Many aminemodified nucleotides are commercially available from, e.g., AppliedBiosystems, Sigma, Jena Bioscience and TriLink. An illustrative, butnon-limiting list of suitable fluors is shown in Table 6.

TABLE 6 Illustrative, but non-limiting fluorophores (fluorescent labels)for use in the primers and/or probes described herein. AbsorbanceEmission Dye Wavelength Wavelength Alexa fluor 345 442 Alexa fluor 430430 545 Alexa fluor 488 494 517 Alexa fluor 532 530 555 Alexa fluor 546556 573 Alexa fluor 555 556 573 Alexa fluor 568 578 603 Alexa fluor 594590 617 Alexa fluor 633 621 639 Alexa fluor 633 650 668 Alexa fluor 660663 690 Alexa fluor 680 679 702 Allophycocyanin 650 660 Aminocoumarin350 445 Cy2 490 510 Cy3 550 570 Cy3.5 581 581 596 Cy5 650 670 Cy5.5 675694 Cy7 743 770 FAM 495 516 Fluorescein FITC 495 518 HEX 535 556Hydroxycoumarin 325 386 Methoxycoumarin 360 410 Red 613 480; 565 613Rhodamine Red-X 560 580 Rox 575 602 R-phycoerythrin (PE) 480; 565 578Tamara 565 580 Texas Red 615 615 TRITC 547 572 TruRed 490; 675 695

If the assay is designed to detect one target DNA sequence then only onefluorescent hybridization probe needs to be used and, in certainembodiments, FAM, TET, or HEX (or one of their alternatives listed inTable 7) will be a good fluorophore to label the probe. Thesefluorophores can readily be excited and detected in variousspectrofluorometric thermal cyclers. In addition, because of theavailability of phosphoramidites derivatives of these fluorophores andthe availability of quencher-linked control—pore glass columns,fluorescent hybridization probes with these labels can be entirelysynthesized in an automated DNA synthesis process, with the advantage ofrelatively less expensive and less labor intensive probe manufacture.

TABLE 7 Additional illustrative fluorophore labels for fluorescenthybridization probes. Excitation Emission Fluorophore AlternativeFluorophore (nm) (nm) Cy3³ NED², Quasar 570¹, Oyster 556⁴ 550 570 Cy5³LC red 670⁵, Quasar 670¹, 650 670 Oyster 645⁴ HEX JOE, VIC ^(B), CALFluor Orange 535 555 560¹ LC red 640⁵ CAL Fluor Red 635 ^(A) 625 640 LCred 705⁵ Cy5.5³ 680 710 ROX LC red 610⁵, CAL Fluor Red 610¹ 575 605 TETCAL Fluor Gold 540¹ 525 540 Texas red LC red 610⁵, CAL Fluor Red 610¹585 605 TMR CAL Fluor Red 590¹ 555 575 ¹CAL and Quasar fluorophores areavailable from Biosearch Technologies; ²VIC and NED are available fromApplied Biosystems; ³Cy dyes are available from Amersham Biosciences;⁴Oyster fluorophores are available from Integrated DNA Technologies; and⁵LC (Light Cycler) fluorophores are available from Roche AppliedScience.

In certain embodiments, multiple target genes are detected in a singlemultiplex reaction. In some embodiments, each probe that is targeted toa different gene is spectrally distinguishable (detectably different)from the other probes utilized in the multiplex reaction. Probecombinations suitable for multiplex detection are known to those ofskill in the art. For example, illustrative combinations of detectablydifferent fluorphores in four target multiplex systems include, but arenot limited to:

1) FAM, TMR, Texas red, and Cy5;

2) FAM, TET, TMR, and Texas Red;

3) FAM, HEX, Texas red, and Cy5; and

4) FAM, Cy3, Texas red, and Cy5.

An illustrative combination of detectably different fluorphores in afive target multiplex systems is FAM, TET, TMR, Texas Red, and Cy5.Illustrative combinations of detectable different fluorophores in a sixtarget multiplex system include, but are not limited to:

1) FAM, TET, HEX, TMR, ROX, and Texas red; and

2) FAM, HEX, LC red 610, LC red 640, LC red 670, and LC red 705.

It will be recognized that these combinations of fluorophores areillustrative and non-limiting and numerous other fluorophores will beavailable to those of skill in the art.

As noted above, for the design of fluorescent hybridization probes thatutilize fluorescence resonance energy transfer (FRET),fluorophore-quencher pairs that have sufficient spectral overlap shouldbe chosen. Fluorophores with an emission maximum between 500 and 550 nm,such as FAM, TET and HEX, are best quenched by quenchers with absorptionmaxima between 450 and 550 nm, such as dabcyl, BHQ-1, and the like (see,e.g., Table 8 for illustrative quencher labels). Fluorophores with anemission maximum above 550 nm, such as rhodamines (including TMR, ROXand Texas red) and Cy dyes (including Cy3 and Cy5) are effectivelyquenched by quenchers with absorption maxima above 550 nm (includingBHQ-2).

For the design of fluorescent hybridization probes that utilize contactquenching, any non-fluorescent quencher can serve as a good acceptor ofenergy from the fluorophore. For example, Cy3 and Cy5 are effectivelyquenched by the BHQ-1 and BHQ-2 quenchers.

TABLE 8 Illustrative quencher labels for fluorescent hybridizationprobes. Absorption Maximum Quencher (nm) BHQ-1⁴ 534 BHQ-2⁴ 580 BHQ-3⁴670 Dabcyl 475 DDQ-I¹ 430 DDQ-II¹ 630 Eclipse² 530 Iowa Black FQ³ 532Iowa Black RQ³ 645 QSY-21⁵ 660 QSY-7⁵ 571 ¹DDQ or Deep Dark Quenchersare available from Eurogentec; ²Eclipse quenchers are available fromEpoch Biosciences; ³Iowa quenchers are available from Integrated DNATechnologies; ⁴BHQ or Black Hole quenchers are available from BiosearchTechnologies; and ⁵QSY quenchers are available from Molecular Probes.

In certain embodiments nucleotides can quench the fluorescence offluorophores, with guanosine being the most efficient quencher, followedby adenosine, cytidine and thymidine. In general, fluorophores with anexcitation wavelength between 500 and 550 nm are quenched moreefficiently by nucleotides than fluorophores with longer excitationwavelengths. In designing fluorescent hybridization probes, it can bedesirable to avoid placing a fluorophore label directly next to aguanosine, to ensure higher fluorescence signals from the fluorophore.

The stabilizing effect of some fluorophore-quencher pairs that interactby contact quenching can have important consequences for the design ofhybridization probes (see, e.g., Marras et al. (2002) Nucleic Acids Res.30: e122; Johansson et al. (2002) J. Am. Chem. Soc. 124: 6950-6956). Forexample, it has been observed that hybridization probes labeled with afluorophore quenched by either BHQ-1 or BHQ-2 show an increase in hybridmelting temperature of about 4° C., compared to hybridization probeswith the same probe sequence, but labeled with fluorophores quenched bydabcyl. It is also noted that strong affinity has been observed betweenthe Cy dyes, Cy3 and Cy5, and the Black Hole quenchers, BHQ-1 and BHQ-2.

In view of the foregoing and the Examples and teachings provided herein,numerous primer/probe combinations will be available for use in themethods and cartridges described herein.

Cartridge, Modules, and Systems for DNA Methylation Analysis.

In certain embodiments cartridges are provided for performing themethods described herein (e.g., determination of DNA methylation and,optionally RNA expression). In certain embodiments the cartridgecomprises a column comprising a first matrix material, a samplereceiving chamber, a temperature controlled channel or chamber, aplurality of chambers containing reagents and/or buffers, and when inuse at least one of said chambers contains a DNA conversion reagent(e.g., DABSO and/or a bisulfite reagent), and at least one of saidchambers contains a desulphonation/elution buffer, and wherein saidcartridge optionally comprises a second column comprising said secondmatrix material. In certain embodiments the cartridge is configured sothat in use, the cartridge comprises a chamber containing a reagentcomprising guanidinium thiocyanate ethanol (GTC-EtOH). In certainembodiments the second column is absent, while in other embodiments thesecond column is present. In certain embodiments the temperaturecontrolled channel or chamber can simply be a heating channel orchamber, or it can be a thermocycling channel or chamber. In certainembodiments the cartridge further comprises a second heating channel orchamber (e.g., a second thermocycling channel or chamber). In certainembodiments the cartridge is configured so that a DNA conversion step(e.g., bisulfite incubation) and/or a desulphonation step occurs in thesame reaction tube or chamber in which one or more PCR reactions arelater performed.

In certain embodiments the bisulfite reagent is provided as a componentof the cartridge. In certain other embodiments the cartridge isconfigured for the bisulfite reagent to be added to the cartridge at ornear the time the sample is placed in the cartridge. In certaininstances, the bisulfite reagent is added directly into a chamber in thecartridge, while in other embodiments, the bisulfite reagent isintroduced into a loading port on the cartridge (e.g., an injectionport) to introduce the bisulfite reagent into the cartridge. In certainembodiments the bisulfite reagent is introduced into the cartridge bythe system operating the cartridge (e.g., a processing module) while thecartridge is operating to determine DNA methylation.

In certain embodiments the reagent comprising guanidinium thiocyanate(e.g., GTC-EtOH) is provided as a component of the cartridge. In certainother embodiments the cartridge is configured for the reagent comprisingguanidinium thiocyanate to be added to the cartridge at or near the timethe sample is placed in the cartridge. In certain instances, the reagentcomprising guanidinium thiocyanate is added directly into a chamber inthe cartridge, while in other embodiments, the reagent comprisingguanidinium thiocyanate is introduced into a loading port on thecartridge (e.g., an injection port) to introduce the bisulfite reagentitno the cartridge. In certain embodiments the reagent comprisingguanidinium thiocyanate is introduced into the cartridge by the systemoperating the cartridge (e.g., a processing module) while the cartridgeis operating to determine DNA methylation.

In various illustrative, but non-limiting embodiments, the conversionreagent (e.g., bisulfite reagent) comprises a compound selected from thegroup consisting of sodium metabisulfite, potassium bisulfite, cesiumbisulfite, DABSO, and ammonium bisulfite. In certain embodiments thebisulfite is provided in a reagent mix comprising scavengers (e.g.,Trolox, hydroquinone, etc.) to prevent sulfite oxidation and/orcatalysts. In certain embodiments the bisulfite is provided in a reagentmix comprising polyamines as catalysts.

In various embodiments the first matrix material and/or said secondmatrix material, when present, comprises a material selected from thegroup consisting of glass or silica, an ion exchange resin, andhydroxyapatite.

In various embodiments the cartridge comprises one or more chambers(e.g., 1 chamber, 2 chambers, 3 chambers, 4 chambers, etc.) eachcontaining one or more reagents selected from the group consisting ofmethylation specific PCR primers, methylation specific PCR probes, PCRenzyme(s) (e.g., polymerase), reverse transcriptase, and PCR reactionbuffer.

In certain embodiments the cartridge contains one or more chamberscontaining primers specific for bisulfite-converted methylated and/orunmethylated sequences. In certain embodiments the cartridge comprisesone or more chambers containing primers and probes for a MethyLight PCRprotocol. In certain embodiments the cartridge comprises one or morechambers containing reagents for TaqMan PCR reactions. In certainembodiments the cartridge comprises one or more chambers containing oneor more fluorescent probes that are markers for amplified methylatedsequences and/or one or more fluorescent probes that are markers foramplified unmethylated sequences. In certain embodiments the probescomprise a fluorescent reporter dye and a quencher dye, where the probesprovides a signal upon cleavage by the 5′ to 3′ nuclease activity of TaqDNA polymerase. In certain embodiments the cartridge comprises aplurality of probes each specific to a different methylated region in anamplified region of interest. In certain embodiments the cartridgecomprises a single probe specific to a methylated region in an amplifiedregion of interest. In certain embodiments the cartridge comprises aplurality of probes each specific to the same methylated region in anamplified region of interest.

Illustrative primers and probes include, but are not limited to primersand/or probes to determine methylation of a promoter region of a geneselected from the group consisting of APC, ARF, CDKN2B, CDKN2A, BRCA1,VLH, hMLH1, MGMT. RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2,TM6SF1, AKR1B1, HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. In certainembodiments the primers and/or probes are selected to determinemethylation of a promoter region of a gene selected from the groupconsisting of MGMT, RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2,TM6SF1, and AKR1B1. In various embodiments the PCR primers, and/orprobes, and/or enzymes are provided as beads, e.g., as described in U.SPatent Publication No: 2006/0068399, which is incorporated herein byreference for the beads and bead formulations described therein.

In various embodiments the cartridge is configured so that the samplereceiving chamber, said column(s), the plurality of chambers, and thetemperature controlled channel or chamber, are selectively in fluidcommunication. In certain embodiments the selective fluid communicationis provided by microfluidic channels and valves. In certain embodimentsthe selective fluid communication is provided by providing the samplereceiving chamber, said column(s), said plurality of chambers, theheating channel or chamber or a port into the heating channel orchamber, disposed around a central valve and selectively in fluidcommunication with a channel in said central valve.

In certain embodiments the cartridge is configured so that, when in use,the cartridge comprises: a first chamber containing a sample; a secondchamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH)solution; a third chamber containing a bisulfite reagent; a fourthchamber containing a buffer; a fifth chamber containing a rinsesolution; and a sixth chamber containing an elution/desulphonationreagent. In certain embodiments the cartridge comprises a seventhchamber containing PCR primers and/or probes and/or PCR enzymes. Incertain embodiments the cartridge comprises an eighth chamber alsocontaining PCR primers and/or probes and/or PCR enzymes.

FIGS. 1A, 1B and 2 illustrate one cartridge suitable for practice of themethods described herein. The illustrated cartridges are based on theGENEXPERT® cartridge (Cepheid, Inc., Sunnyvale, Calif.). As shown inFIG. 2, panel A the cartridge 200 comprises a cartridge body 202containing a plurality of reagent and/or buffer chambers 208. Thechambers are disposed around a central syringe barrel 206 that is influid communication with a valve body 210 (panel B and FIG. 1B) and thatis sealed with a gasket 204. The valve body 210 can comprise a cap 212and the entire cartridge body can be supported on a cartridge base 226.A “plunger” not shown can be operated to draw fluid into the syringebarrel 206 and rotation of the syringe barrel 206 and asscoated valvebody 212 provides selective fluid communication between the chambers 208a cavity 214 that can contain a matrix material as described herein andfunction as a column. In various embodiments the cartridge furthercomprises one or more temperature controlled channels or chambers 216that can, in certain embodiments, function as thermocycling chambers.The temperature controlled channels or chambers are also selectively influid communication with the cavity 214 and/or the chambers 208. Asshown in FIG. 1A, in certain embodiments, the cartridge provides opticalwindows to provide real-time detection of, e.g., amplification products,base identity in sequencing operations, and the like.

In certain embodiments the cartridge 200 is configured for insertioninto a reaction module 300, e.g., as shown in FIG. 3A. As illustrated inFIG. 3B the module is configured to receive the cartridge 200. Incertain embodiments the reaction module provides heating plates 308 toheat the temperature controlled chamber or channel. The module canoptionally additionally include a fan 304 to provide cooling where thetemperature controlled channel or chamber is a thermocycling channel orchamber. Electronic circuitry 302 can be provided to pass information(e.g., optical information) top a computer for analysis. In certainembodiments the module can contain optical blocks 306 to provideexcitation and/or detection of one or more (e.g., 1, 2, 3, 4, or more)optical signals representing, e.g., various nucleic acid targets. Invarious embodiments an electrical connector 312 can be provided forinterfacing the module with a system (e.g. system controller or with adiscrete analysis/controller unit. As illustrated, in FIG. 3B the samplecan be introduced into the cartridge using a pipette 310.

In certain embodiments, the module also contains a controller thatoperates a plunger in the syringe barrel and the rotation of the valvebody.

In certain embodiments a system (e.g., a processing unit) is provided.One illustrative, but non-limiting embodiment is shown in FIG. 3C. Incertain embodiments, the processing unit comprises an enclosureconfigured to contain one or more sample processing modules where eachprocessing module is configured to hold and operate a removablecartridge as described herein. In certain embodiments the system isconfigured to operate the sample processing modules to perform sampleprocessing to determine methylation of one or more target nucleic acidsand optionally to determine the level of one or more target RNA/DNAsequences within a corresponding removable sample cartridge, wherein theprocessing on a sample within the corresponding removable samplecartridge performs a method as described herein. In certain embodimentsthe system is configured to contain one sample processing module. Incertain embodiments the system is configured to contain at least twosample processing modules, or at least 4 sample processing modules, orat least 8 sample processing modules, or at least 12 sample processingmodules, or at least 16 sample processing modules, or at least 20 sampleprocessing modules, or at least 24 sample processing modules, or atleast 28 sample processing modules, or at least 32 sample processingmodules, or at least 64 sample processing modules, or at least 128sample processing modules. In certain embodiments the system provides auser interface that allows the user input operational instructionsand/or to monitor operation of the cartridges to determine DNAmethylation.

While the methods described herein are described primarily withreference to the GENEXPERT® cartridge by Cepheid Inc. (Sunnyvale,Calif.) (see, e.g., FIG. 1A), it will be recognized, that in view of theteachings provided herein the methods can be implemented on othercartridge/microfluidic systems. Such cartridge/microfluidic systems caninclude, for example microfluidic systems implemented using softlithography, micro/nano-fabricated microfluidic systems implementedusing hard lithography, and the like.

High Volume Sample Preparation (HVSP) Cartridge.

In various embodiments cartridges are provided for the preparation oflarge sample volumes. In certain embodiments the sample preparationcartridges comprises GENEXPERT® cartridges modified for high volumesample preparation (e.g., as shown in FIG. 20). In certain embodiments,e.g., when the cartridge is based on a GENEXPERT® cartridge comprisesone or more channels or chambers comprising an affinity matrix thatbinds DNA, a plurality of chambers disposed around a central valveassembly and selectively in fluid communication with said central valveassembly where the central valve assembly is configured to accommodate aplunger that is capable of drawing fluid into or out of a chamber influid communication with the central valve wherein said plurality ofchambers comprises at least two different chambers each configured toreceive up to about 4 ml (or up to about 5 ml) of sample solution (incertain embodiments chamber 2 has a maximum volume of about 4 ml, whilechamber 3 has a maximum volume of about 4.5 ml), a chamber containingPEG (e.g., PEG200), a chamber containing an alkaline solution (e.g., KOHsolution), and a chamber containing a buffer (e.g., Tris). In certainembodiments the plurality of chambers comprises at least three differentchambers each configured to receive up to about 4 ml (or up to about 5ml) of sample solution. In certain embodiments the plurality of chamberscomprises a chamber containing a wash solution (e.g., GTC-ethanol washsolution which is typically 1.25M guanidinium thiocyanate, 25 mM Tris pH7.0, 50% ethanol). In certain embodiments the cartridge comprises achamber configured for removal of a processed sample. In certainembodiments the sample chambers, when in use, contain sample solution,GTC and alcohol (e.g., isopropanol). In certain embodiments the samplechambers, when in use contain sample solution, GTC and alcohol insubstantially equal volumes. In certain embodiments the cartridge, whenin use, comprises 4 ml of sample solution GTC and isopropanol disposedin each of said chambers configured to receive a sample. In certainembodiments the cartridge provides DNA or RNA recovery that issubstantially linear with respect to the sample volume between 0.5 mland about 4 ml of sample.

In certain embodiments the HVSP cartridge is configured to perform a DNAconversion (e.g., bisulfite conversion) to provide a methylationanalysis. Accordingly in certain embodiments, the HVSP cartridge isconfigured to contain, or to receive immediately or shortly prior touse, a conversion reagent (e.g. a bisulfite reagent, DABSO, etc.). Incertain embodiments, the HVSP cartridge can be configured to alsocontain reagents for and to provide a desulphonation of converted DNA.Alternatively, in certain embodiments, the conversion is performed inthe HSVP cartridge while the desulphonation and methylation analysis(e.g., PCR) is performed in the second cartridge (e.g., as illustratedin the work flows shown in FIG. 20B).

Two-Cartridge Methylation Analysis

In various embodiments the methylation analysis methods described hereinare performed using a two-cartridge system (a set of two cartridges suchas a “first” cartridge (e.g., a high volume sample preparationcartridge), and a “second” cartridge (e.g., a qPCR cartridge)), e.g., asillustrated in FIG. 20B. As illustrated in this figure, in certainembodiments, sample preparation and bisulfite conversion can beperformed in a first cartridge. The converted DNA is then transferred tosecond cartridge wherein it is washed and desulphonated. In certainembodiments the second cartridge is additionally utilized to analyze theconverted (or unconverted) DNA, e.g., via PCR (e.g.,methylation-specific PCR).

In certain embodiments the first cartridge can be a high-volume samplecartridge as described above where the sample preparation cartridgecontains a bisulfite conversion reagent as described herein. In certainembodiments the high-volume cartridge provides a larger sample chamberor multiple sample chambers to permit cleanup and conversion of largeramounts of DNA.

In certain embodiments the first cartridge of the two-cartridge setcomprises a sample receiving chamber, a “column” comprising a firstmatrix material, a temperature controlled channel or chamber, a sampleremoval chamber, and a plurality of chambers containing reagents and/orbuffers. Typically, in use, at least one of the chambers contains abisulfite reagent (e.g., as described herein). In certain embodimentsthe second cartridge of the two-cartridge set comprises a samplereceiving chamber, a “column” comprising a matrix material, atemperature controlled channel or chamber, a plurality of chamberscontaining reagents and/or buffers. Typically, in use, at least one ofthe plurality of chambers in the second cartridge contains adesulphonation and/or elution reagent. In certain embodiments the secondcartridge contains reagents for PCR amplification (e.g., methylationspecific PCR) and the detection of an amplification product.

In certain embodiments illustrative, but non-limiting embodiments, thefirst cartridge is used for sample preparation (e.g., binding of DNA toa first matrix material (e.g., glass fiber), washing and eluting of thebound DNA) and bisulfite conversion of the washed DNA. In certainembodiments the bisulfite conversion comprise combining the washed DNAwith a bisulfite reagent and heating the mixture in thetemperature-controlled channel or chamber. The resulting converted DNAis then mixed with a wash buffer (e.g., 1.25M GTC, 25 mM Tris pH 7.0,50% ethanol) and moved into a sample removal chamber (e.g., chamber 2)where it can be removed (e.g., removed using a pipette, a syringe orsyringe, pump, and the like).

In certain embodiments the bisulfite converted DNA in wash buffer isintroduced into a sample receiving chamber of the second cartridge. Thesecond cartridge is operated to bind the DNA to a second matrix material(e.g., glass fiber “column”), to wash and elute the bound DNA and todesulphonate the DNA. In certain embodiments the DNA is desulphonated onthe column or as it is eluted from the second matrix material. Incertain embodiments the DNA is desulphonated after removal from thesecond matrix material. In certain embodiments the desulphonated DNA isthen mixed, e.g., with enzyme and primer/probe beads for PCR in thesecond cartridge. In certain embodiments the desulphonated DNA isremoved and can be introduced into a third cartridge or other reactionsystem for further analysis (e.g., sequencing).

In certain embodiments, in the first cartridge, the sample receivingchamber, column, plurality of chambers, sample removal chamber, andtemperature-controlled heating channel or chamber, are selectively influid communication (e.g., by microfluidic channels and/or valves);and/or, in the second cartridge, the sample receiving chamber, thecolumn, the plurality of chambers, and the temperature-controlledheating channel or chamber, are selectively in fluid communication(e.g., by microfluidic channels and/or valves).

In certain embodiments, in the first cartridge, the sample receivingchamber, the column, the plurality of chambers, the sample removalchamber, and the temperature controlled channel or chamber or a portinto the temperature controlled channel or chamber, are disposed arounda central valve and selectively in fluid communication with one or morechannels in the central valve. The central valve can be configured toaccommodate a plunger that is capable of drawing fluid into or out of achamber(s) in fluid communication with the central valve. In certainembodiments, in the second cartridge, the sample receiving chamber, thecolumn, the plurality of chambers, and the temperature controlledchannel or chamber or a port into the temperature controlled channel orchamber, are disposed around a central valve and selectively in fluidcommunication with one or more channels in the central valve. In certainembodiments the central valve is configured to accommodate a plungerthat is capable of drawing fluid into or out of one or more chambers influid communication with the central valve.

In certain embodiments the first cartridge and/or the second cartridgeis a GENEXPERT® cartridge, or a modified GENEXPERT® cartridge (e.g., asillustrated in FIGS. 1A, 1B, 2, 13A, 17, and 20A). In certain,illustrative, but non-limiting embodiments, the first cartridge isconfigured as shown in Table 9. In certain embodiments the chambernumbering corresponds to the chambers illustrated in FIG. 1B.

TABLE 9 One illustrative, but non-limiting configuration of a “first”cartridge for DNA preparation and bisulfite conversion. Chamber #Reagent Volume Chamber 1 Empty N/A Chamber 2 Sample in GTC Tween 2.5 mLChamber 3 1.25M GTC, 25 mM Tris pH 3.0 mL 7.0, 50% Ethanol Chamber 4 NH4Bisulfite ~150 μL Chamber 5 Tris pH 8.5 2.0 mL Chamber 6 Empty N/AChamber 7 Empty N/A Chamber 8 PEG200 1.0 mL Chamber 9 Empty N/A Chamber10 15 mM KOH 500 μL Chamber 11 Empty N/A

In certain, illustrative, but non-limiting embodiments, the secondcartridge is configured as shown in Table 10. In certain embodiments thechamber numbering corresponds to the chambers illustrated in FIG. 1B.

TABLE 10 One illustrative, but non-limiting configuration of a “second”cartridge for desulphonation of converted DNA and optional PCR of thedesulphonated product. Chamber # Reagent Volume Chamber 1 Empty N/AChamber 2 1.25M GTC, 25 mM Tris 2.0 mL pH 7.0, 50% Ethanol Chamber 3with funnel Sample from 1st Cartridge ~1.6 mL Chamber 4 Empty N/AChamber 5 Tris pH 8.5 2.0 mL Chamber 6 Empty N/A Chamber 7 Empty N/AChamber 8 PEG200 1.0 mL Chamber 9 Enzyme Bead, 1x Internal 2x Beads BeadChamber 10 15 mM KOH 500 μL Chamber 11 Tris Bead, Enzyme Bead, 3x Beads1x External Bead

In certain embodiments the second cartridge is configured to use one ormore of the following genes as control genes: MYOD1, COL2A1, NONO,and/or TUBB.

In certain embodiments using the two-cartridge format is possible, interalia, to run a non-nested PCR reaction or a methylation specific preampin the 2^(nd) cartridge.

The foregoing configurations are illustrative and non-limiting. Usingthe teaching provided herein numerous other two-cartridge formats willbe available to one of skill in the art.

By way of illustration, data showing the results of a two-cartridgeanalysis of urine and sputum samples are shown in FIGS. 35, 36, and 37.In particular, FIG. 35 shows a two-cartridge methylation analysis ofBNC1 and ACTB. 0, 25, 50, and 100 copies of 100% methylated control DNAwas spiked into serum and processed in the 1st sample prep cartridge(see, e.g., Table 9), and then qPCR was run on the extracted, convertedDNA sample in the second cartridge (see, e.g., Table 10) containingprimer and probe sets for the methylated BNC1 promoter and a methylationindependent primer and probe set for the ACTB promoter.

FIG. 36 shows the results of bisulfite conversion analysis of normalurine samples. 2.0 mL of normal urine was mixed with 1.5 mL of LysisBuffer [4.5M GTC, 1% Tween20] and 0.5 mL of Ethanol in Condition #6. 1.5mL of normal urine was mixed with 1.25 mL of Lysis Buffer and 1.25 mL ofEthanol in Condition #7. The 4.0 mL samples were pipetted into Chamber 2of the methylation analysis cartridge 2 and analyzed for converted ACTB(right) and unconverted HMBS (left).

FIG. 37 shows the results of methylation analysis of normal and cancersputum samples. Seven sputum samples were tested for SOX17, TAC1, andHOXA7 methylation using the GENEXPERT® methylation cartridge (C) and acomparative assay (M). Both raw Ct's and a deltaCt (to ACTB) arereported in this figure.

The foregoing configurations are illustrative and non-limiting. Usingthe teaching provided herein numerous other two-cartridge formats willbe available to one of skill in the art.

cfDNA Sample Preparation Cartridge.

In certain embodiments a sample preparation cartridge is provided thatis articular well suited to the preparation (and optional analysis) ofnucleic acids from plasma or serum is provide. One illustrative, butnon-limiting embodiment is shown in FIG. 17. As illustrated therein incertain embodiments the cartridge comprises a channel or chambercomprising an affinity matrix that binds DNA, a plurality of chambersdisposed around a central valve assembly and selectively in fluidcommunication with the central valve assembly where the central valveassembly is configured to accommodate a plunger that is capable ofdrawing fluid into or out of a chamber in fluid communication with thecentral valve where the plurality of chambers comprises: a chamberconfigured to receive up to about 5 ml or up to about 4 ml of samplesolution; a chamber containing PEG (e.g., PEG200); a chamber containingGTC-EtOH; a chamber containing an alkaline solution (e.g., KOH); and achamber containing a buffer (e.g., Tris). In certain embodiments theplurality of chambers further comprises a chamber containing aconversion reagent (e.g., a bisulfite reagent). In certain embodimentsthe plurality of chambers comprises a chamber containing a wash solution(e.g., GTC-ethanol wash (typically 1.25M guanidinium thiocyanate, 25 mMTris pH 7.0, 50% ethanol)). In certain embodiments the plurality ofchambers comprises a chamber containing beads comprising one or more PCRprimers and/or probes. In certain embodiments the chamber containing PEGcontains about 1 ml of PEG. In certain embodiments the chambercontaining an alkaline solution contains about 500 μL of solution. Incertain embodiments the chamber containing GTC-EtOH contains about 2 mlGTC-EtOH. In certain embodiments the chamber containing a buffercontains about 2 mL of buffer.

It will be recognized that this configuration is illustrative, and usingthe teaching provided herein numerous other preparation cartridgeconfigurations will be available to one of skill in the art.

Use of DABSO as an Alternative to Bisulfite

It was a surprising discovery that DABSO can be used to perform aconversion of DNA in a manner analogous to the use of bisulfites for theconversion of DNA and detection of methylation. Accordingly, in certainembodiments, methods of utilizing DABSO to converting cytosine residuesin a DNA to uracil, while leaving 5-methylcytosine residuessubstantially unaffected are provided. In certain embodiments themethods involve contacting a sample comprising DNA with DABSO to convertthe DNA, and desulphonating the converted DNA, to produce a DNA in whichcytosine residues are converted to uracil, but 5-methylcytosine residuessubstantially unaffected. In certain embodiments the DABSO is providedat a concentration ranging from about 2 M up to about 5 M. In certainembodiments the DABSO is provided at a concentration of about 2.5 M. Incertain embodiments the DABSO is dissolved in an alkaline aqueoussolution (e.g., a KOH solution). In certain embodiments the reagentcomprising DABSO comprises DABSO dissolved in a solution comprising KOH.

In certain embodiments the methods involve heating the DABSO/DNAsolution to a temperature ranging from about 55° C. to about 90° C. Incertain embodiments the DABSO is reacted with the DNA for a period oftime ranging from about 15 minutes up to about 90 minutes. After the DNAis converted, it is desulphonated (e.g., by contacting the converted DNAwith an alkaline reagent (e.g., KOH solution). In certain embodimentsthe conversion and/or desulphonation is performed on the DNA bound to acolumn, while in other embodiments the conversion and/or desulphonationis performed on the DNA in solution.

Also provided are methods of analyzing DNA methylation, where themethods involve providing a DNA sample, converting DNA in the sampleusing a DABSO reagent, e.g., as described above, and performingmethylation specific PCR and/or nucleic acid sequencing, and/or highresolution melting analysis (HRM) on the converted nucleic acid todetermine the methylation of said nucleic acid. In certain embodimentsthe providing of a DNA sample comprises preparing a sample as describedherein (e.g., using lysis solutions and/or preparation cartridges asdescribed herein.

Kits.

Kits for Methylation Detection.

In certain embodiments kits are provided for performing the methodsdescribed herein. In one illustrative embodiment, the kits comprise acontainer containing a reaction cartridge as described herein, acontainer containing a sample processing reagent as described herein,and a container containing a conversion reagent (e.g., a bisulfitereagent) as described herein. In certain embodiments the bisulfitereagent is provided in a chamber of the cartridge. In certainembodiments the bisulfite reagent is provided in a container separatefrom the cartridge. In certain embodiments, the sample processingreagent is provided in a chamber of the cartridge. In certainembodiments, particularly where the sample processing reagent comprisesguanidinium thiocyanate the sample processing reagent is provided in acontainer separate from the cartridge.

In certain embodiments the kits can contain the cartridges for atwo-cartridge set as described herein. Thus, in certain embodiments thekits can contain a “first” cartridge that is a sample preparationcartridge configured to prepare a sample and perform a bisulfiteconversion of DNA in the sample (see, e.g., FIG. 20B, left panel) and a“second” cartridge configured to desulphonated the converted DNA and, incertain embodiments, perform a subsequent analysis (e.g.,methylation-specific PCR) (see, e.g., FIG. 20B, right panel). In certainembodiments the first cartridge and the second cartridge are provided inseparate containers in the kit, while in other embodiments, the firstcartridge and second cartridge are provided in the same container. Incertain embodiments the two-cartridge kits can contain devices (e.g.,funnels, pipette tips, etc.) to facilitate transfer of a sample from thesample removal chamber of the first cartridge to the sample receivingchamber of the second cartridge.

In addition, the kits optionally include labeling and/or instructionalmaterials providing directions (i.e., protocols) for the use of thecartridges described herein to determine DNA methylation and,optionally, RNA expression.

In certain embodiments a kit for the determination of DNA methylation isprovided where the kit comprises a container containing a cartridge fordetermining the methylation state of a nucleic acid as described herein.In certain embodiments the kit further comprises a lysis solution asdescribed herein (e.g., a lysis solution for serum or plasma, e.g., asdescribed in Table 13, and/or a lysis solution for FFPE samples, e.g.,as described in Table 14). In certain embodiments the kit comprises acontainer containing proteinase K. In certain embodiments the kitcontains a conversion reagent (e.g., a bisulfite reagent) in thecartridge or in a container separate from the cartridge. In certainembodiments the separate container can contain a pre-measured volume ofconversion reagent suitable for one “run” of the cartridge. In certainembodiments the conversion reagent comprises a compound selected fromthe group consisting of sodium metabisulfite, potassium bisulfite,cesium bisulfite, ammonium bisulfite, and DABSO. In certain embodimentsthe kit comprises a container containing a sample processing reagent. Incertain embodiments the sample processing reagent comprises guanidiumthiocyanate and/or ethanol.

In various embodiments the kit can additionally contain a cartridge forsample preparation as described herein (e.g., as illustrated in FIG.20).

In certain embodiments the kit contains instructional materials teachingthe use of a cartridge for the determination of DNA methylation. Where asample preparation cartridge is included in the kit the kit canadditionally contain instructional materials teaching the use andoperation of the sample preparation cartridge.

Kits for DABSO DNA Conversion and Methylation Detection.

In certain embodiments kits are provided for the use of DABSO as aconversion reagent, e.g., in the detection of the methylation state of aDNA. In certain embodiments the kits comprise a container containing aconversion reagent comprising DABSO, and a container containing adesulphonation reagent. In certain embodiments the kit comprises acolumn comprising an affinity matrix (e.g., a silica matrix material).In certain embodiments the kits comprise a container containing abinding buffer and/or a container containing an elution buffer. Incertain embodiments the kit comprises a container containing a washbuffer.

In certain embodiments the kit further comprises a lysis solution asdescribed herein (e.g., a lysis solution for serum or plasma, e.g., asdescribed in Table 13, and/or a lysis solution for FFPE samples, e.g.,as described in Table 14). In certain embodiments the kit comprises acontainer containing proteinase K.

In various embodiments the kit can additionally contain a cartridge forsample preparation as described herein (e.g., as illustrated in FIG.20).

In certain embodiments the kit contains instructional materials teachingthe use of the kit to convert a nuclei acid for determination of themethylation state of the nucleic acid.

While the instructional materials in the kits described above typicallycomprise written or printed materials they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated by this invention. Such media include, but arenot limited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

To validate the method human genomic DNA (HGDNA) was used as a startingsample to monitor sample preparation, bisulfite conversion, samplecleanup, and methylation specific qPCR in a Cepheid GENEXPERT®cartridge. In order to measure bisulfite conversion efficiency, half ofthe DNA-bisulfite mix was loaded and heated in the 50 μL cartridge tubeduring the bisulfite conversion step. Therefore, under optimalconversion conditions approximately half of the HGDNA is converted andthe other half remains unconverted.

Primers and Taqman probes for the qPCR step were designed for oneunconverted gene (HMBS (hydroxymethylbilane synthase housekeeping gene))and one converted gene (ACTB (beta actin)), and the conversionefficiency was then quantitated by comparison of cycle threshold values(Cts). Both ACTB and HMBS are commonly used as single or low copyreference genes, and thus we expect similar copy numbers per ng ofHGDNA.

A representative GENEXPERT® run from 300 ng of HGDNA is shown below inFIG. 5, with the ACTB qPCR curve in green and the HMBS qPCR curve inblue. The qPCR reaction was run for 45 cycles with a 3 temperature cycleof 96° C. for 5 seconds, 60° C. for 15 seconds, and 72° C. for 15seconds. At a manual threshold setting of 20 fluorescence units weobserved a Ct of 31.7 for the converted ACTB gene and a Ct of 32.7 forthe unconverted HMBS gene. Importantly, this result demonstrates that weare able to achieve near-optimal bisulfite conversion efficiency ofHGDNA in our cartridge at physiological relevant concentrations of DNAfound in FFPE tissue slices and plasma/serum samples. Furtherspecificity for fully converted sequences can be achieved through anested qPCR reaction or by heating the entire sample. However, neitheroption would be absolutely required for methylation specific qPCR in theGENEXPERT® because primer and probe sets are designed to amplify onlythe converted sequences. Thus remaining unconverted DNA sequences wouldact as carrier DNA, which notably is frequently added during bisulfiteconversion, DNA isolation, and PCR methods.

Example 2

FIGS. 6A and 6B show the linearity of converted ACTB. In particular,FIG. 6A shows the results of a 15 cycle nested qPCR for ACTB usinghgDNA. As can be seen from the panel on the right the signal (Ct value)is substantially linear between about 25,000 copies and about 100copies. FIG. 6B shows the results of a 20 cycle nested qPCR for ACTBusing hgDNA. These plots demonstrate the sensitivity of the cartridgefor hgDNA. Dropouts start occurring around 20-50 copies with asensitivity of about 25 copies of converted DNA.

FIGS. 7A, 7B, and 7C show the results of qPCR for six methylated targets(AKR1B1, HOXB4, TM6SF1, RASGRF2, and RASSF1A). FIG. 7A show the resultsof 20 cycle nested qPCR for controls (25 ng of HSDNA, and 5000 MBA-453cells whose DNA is not bisulfite-converted). FIG. 7B shows the resultsof 20 cycle nested qPCR for the six methylated targets using DNA fromMBA-453 cells that has been bisulfite converted. A strong signal isshown for all targets. HIST1H3C was not reliably detected. FIG. 7C showsthe results of 20 cycle nested qPCR for the six methylated targets usingDNA from MBA-453 cells that has been bisulfite converted and is in acarrier comprising 1 μg of SS and 10 ng of HS DNA. Dropouts wereobserved at about 100 cells and below, however, with the carrier, therewere significantly fewer dropouts.

Example 3

FIG. 8 illustrates the results of a determination of conversionefficiency. The conversion efficiency is about 66% (˜1 Ct) thedifference between unconverted HMBS and converted ACTB. Ideal Ct with100% binding/elution, 100% conversion, and 100% binding elution is about24-25. The experiments appear to show a 50% binding/elution, 50-66%conversion, and 50% binding/elution for a 10-fold reduction and a Ct ofabout 27.

FIG. 9 illustrates the increase in specificity for converted DNAproduced by nested qPCR. Nested PCR appears to increase the specificityfor converted DNA, to increase the specificity for methylated DNA and toreduce contamination issues.

FIG. 10 illustrates the specificity of the methylation cartridge. Nospecificity is shown for unconverted DNA (top panel) or unmethylated DNA(bottom panel) except for HIST1H3C.

FIGS. 11A and 11B show some illustrative but non-limiting workflows foranalysis of methylation using a cartridge (e.g., a GENEXPERT®cartridge). FIG. 11A illustrates one work flow for analysis of DNAmethylation in a serum sample. As illustrated in this workflow, serum isadded to a lysis reagent vial and mixed/vortexed. The sample is thendispensed into a sample port in the cartridge. The cartridge is placedin the system for analysis.

FIG. 11A illustrates one work flow for analysis of DNA methylation in atissue section (e.g., frozen or formalin-fixed paraffin embedded (FFPE)section). As shown therein, in one embodiment, a tissue section (e.g., a4 μm FFPE section) is provided. FFPE lysis reagents are added (see,e.g., PCT/US2013/061863 (WO/2014/052551 for illustrative lysis reagents)and the mixture can be heated. Ethanol can be added and the mixturevortexed. The sample is then dispensed into a sample port in thecartridge. The cartridge is placed in the system for analysis.

FIG. 12 illustrates the results for a FFPE cell button for converted ALUand methylated RASSF1A.

Example 4 Detection of Markers for Breast Cancer Monitoring Materialsand Methods:

Either 1000 MBA-453 cells or 25 ng of human sperm (HS) DNA were added to2.5 mL of binding buffer (2.25 M Guanidinium thiocyanate, 22.5 mM TrispH 7.0, 0.5% Tween20, 50% Ethanol, and 0.005% SE-15 antifoam). The 2.5mL solution of cells or DNA was added to chamber 2 of the Cepheidmethylation cartridge (layout in FIG. 13A). The remaining chambers inthe methylation cartridge were filled as follows: Chamber 3—3.2 mL ofWash buffer (1.25M Guanidinium thiocyanate, 25 mM Tris pH 7.0, 50%Ethanol), Chamber 4—90 μL of 7M Ammonium Bisulfite, Chamber 5-4 mL of 50mM Tris pH 8.5, Chamber 8-1 mL of PEG200 Rinse, Chamber 9—quantitativePCR beads including EZR (Taq) and TSR (6 target breast cancer multiplexfor RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, TM6SF1, see Table 11,below), Chamber 10—500 μL of 15 mM KOH, and Chamber 11—nested beadsincluding EZR (Taq) and TSR (6 target breast cancer multiplex forRASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, TM6SF1). The methylationcartridge was then loaded into a Cepheid GeneXpert and the entirety ofthe methylation assay was completed by the GeneXpert—the first DNAsample prep, the bisulfite conversion, the second post conversion DNAsample prep, the desulphonation, and the 20 cycle nested andquantitative PCR reactions.

A flow chart illustrating the methylation protocol is shown in FIG. 13B.It is noted that the PEG200 was filled in the waste chamber 8, and afterthe assay starts the PEG200 is dispensed into Chamber 1. The PEG200 is aviscous liquid that cannot easily be directly loaded in the smallerchamber 1. Additionally, chamber 1 acts as an air chamber when thecartridge is first loaded before becoming the PEG200 chamber. Thus, theassay begins with Chamber 1=air and Chamber 8=PEG200 and is quicklyswitched to Chamber 1=PEG200 and Chamber 8=Waste after cartridgeloading.

The numbers shown in the “Initial Vol.” column of FIG. 13A just refer toliquid volumes. In this case there are just 2× beads in chamber 11-1×TSRbead (primer and probes for the 6 targets) and 1×EZR bead (Phoenix Taq).These beads are for the final qPCR reaction. Similarly, there are 3×beads in chamber 9-1×TSR bead (primers for the 6 targets), 1×Tris bead(to quench KOH) and 1×EZR bead (Phoenix Taq). These beads are for thefirst 15-20 cycle PCR reaction.

It is also noted that Chamber 6 is an air chamber throughout the entireassay and is never filled. Chamber 7 is used as sort of a gateway to thePCR tube in the back of the cartridge. It is not filled to start theassay but is filled during the assay on 3 occasions before loading intothe tube—1) the DNA-bisulfite mix that is heated in the tube forconversion 2) the 15-20 cycle PCR reaction and 3) the final qPCRreaction.

The primers shown in the Table 11 provided shows five sequences for eachgene—two extension primers and 2 qPCR primers for each nestedamplification and one probe. The first 15-20 cycle PCR reaction was notspecific for methylation but only the converted DNA sequences (i.e.,they do not cross CpGs and in a couple instances when they do we use anR=purine or Y=pyrimidine to catch both methylated and unmethylated). Thesecond 45 cycle qPCR reaction contains both primers and probes that arespecific for typically 2-3 methylated CpGs.

Results:

The methylation cartridge was run using 1000 MBA-453 cells with andwithout bisulfite (FIG. 15A-15B) and 25 ng of HS DNA with bisulfite(FIG. 15C) that was primarily unmethylated at each gene promoter withthe exception of HIST1H3C. There was little or no amplification of anyof the targets in either the no bisulfite or unmethylated HS DNA controlreactions (FIG. 15A, 15C). With the addition of bisulfite, themethylation cartridge picked up high levels of methylation at multiplegene promoters from 1000 MBA-453 cells, specifically AKR1B1, RASSF1A,HOXB4, and RASGRF2.

TABLE 11 Nested primers for RASSF1A,AKR1B1, HOXB4, HIST1H3C, RASGRF2, and TM6SF1. Gene/ SEQ Probe ID nameType SEQUENCE NO RASSF1A olAK61 ext GTTTTATAGTT(T*)TTGTATTTAGG  62primer olAK41 ext AACTCAATAAACTCAAACTCCC  42 primer olAK1 qPCRGCGTTGAAGTCGGGGTTC   2 primer olAK2 qPCR CCCGTACTTCGCTAACTTTAAACG   3primer olAK63 qPCR fluor-(C*)TGGTTTCGT(T-  64 probequencher)CGGT(T*)CGCG- quencher/blocker HIST1H3C olAK60 extGGATTTTTGAAATATTATAGGATT  61 primer AATTAG olAK43 extATAAAATTTCTTCACRCCACC  44 primer olAK59 qPCR TCGTACGAAGTAAATAGTTCGTAAG 60 primer olAK54 qPCR CCGATAACCGAAACGCTCTTAC  56 primer olAK65 qPCRfluor-  66 probe CAAACTACTTACGCGAAACTT(T*) ACCGCC-quencher/blockerRASGRF2 olAK44 ext GAGGGAGTTAGTTGGGTTAT  45 primer olAK45 extCCTCCAAAAAATACATACCC  46 primer olAK13 qPCR GTAAGAAGACGGTCGAGGCG  14primer olAK14 qPCR ACAACTCTACTCGCCCTCGAA  15 primer olAK67 qPCR fluor-129 probe AAACGAACCACTTCTCG(T*)ACCA ACGAC-quencher/blocker AKR1B1 olAK58ext GYGTAATTAAT(T*)AGAAGGTTTTTT  59 olAK47 ext AACACCTACCTTCCAAATAC  48olAK19 qPCR GCGCGTTAATCGTAGGCGTTT  20 olAK20 qPCR CCCAATACGATACGACCTTAAC 12 olAK75 qPCR fluor-(C*)A(C*)GCGTACCT(T-  76quencher)TAAA(T*)AACCCG(T*) AAAATCG-quencher/blocker HOXB4 olAK48 extTTAGAGGYGAGAGAGTAGTT  49 primer olAK49 ext AAACTACTACTAACCRCCTC  50primer olAK25 qPCR CGGGATTTTGGGTTTTCGTCG  26 primer olAK26 qPCRCGACGAATAACGACGCAAAAAC  27 primer olAK76 qPCR fluor-  77 probeAACCGAACGA(T*)AACGAAA(N*) ACGACGAA-quencher/blocker TM6SF1 olAK50 extAGGAGATATYGTTGAGGGGA  51 primer olAK51 ext TCACTCATACTAAACCRCCAA  52primer olAK56 qPCR GTTTAGCGGGATGCGGTG  57 primer olAK57 qPCRACACGAAAACCCCGATAAC  58 primer olAK77 qPCR fluor-AAACACTCATCGCAACCGC  34probe CGCG-quencher/blocker C*, T* are optionally functionalized (e.g.,to alter probe T_(m)) bases.

The primers shown in Table 11 are illustrative and not limiting.Numerous other primers and nested primer sets will be available to thoseof skill in the art. By way of example, illustrative primers for thedetection of methylation of ADAMTS1 and BNC1 genes associated withpancreatic cancer and for the detection of methylation of the MGMT geneassociated with glioma are shown in Table 12.

TABLE 12 Illustrative primers for the detectionof methylation of ADAMTS1 and BNC1 genesassociated with pancreatic cancer and for thedetection of methylation of the MGMT gene associated with glioma. Gene/SEQ Probe ID name Type SEQUENCE NO BNC1 ext CCCRCAAACCRCGAAAACCTC 227ext CCGACGACCGACG 235 qPCR GTTTTTTTTYGGGAGAGGTAAATA 228 qPCRGGGAGAGGTAAATATCGATAC 236 qPCR fluor-TGGYGGGGG(T*)AGA(T*) 389ATTTT-quencher/blocker ADAMTS1 ext CRCCTCCRAAACTAAAACAAC 229 extCGCGAAAATTAATACCTAACG 237 qPCR GGGTTATTGTAAAGTTAGGGTG 230 qPCRTTAGGGTGCGTTATCGGAC 238 qPCR fluor-TCTACTCAAAACTCTCCCCT 390CTCC-quencher/blocker MGMT ext1 GTTTT(T*)AGAAYG(T*)TTTGYGTTT 263 ext2AAAAAAC(T*)CCRCACTCTTCC 265 qPCR TTTCGACGTTCGTAGGTTTTCGC 266 qPCRGCACTCTTCCGAAAACGAAACG 267 qPCR fluor- 268 probeCCAAACAC(T*)CACCAAATC(N*)CAA AC-quencher/blocker

Example 5 Sample Preparation for Plasma and FFPE Samples

FIG. 17 illustrates one configuration of a cartridge that can be used toprepare DNA samples for PCR and/or methylation detection. The sample,obtained from serum or plasma, or an FFPE sample can simply beintroduced into a sample chamber of the cartridge (e.g., chamber 3) andoperation of the cartridge as described herein provides a sample readyfor PCR and/or methylation detection.

Sample Preparation

In one illustrative, but non limiting embodiment, a serum or plasmasample is prepared (e.g., for analysis of cfDNA) by treating the serumor plasma with proteinase K. Then the proteinase K treated serum/plasmais mixed with a lysis solution comprising guanidinium thiocyanate (GTC),buffer (e.g., Tris pH 7.0), a detergent (e.g., Tween 20), and anoptional antifoam (e.g., antifoam SE15). An alcohol (e.g., isopropanol)is added to the solution which is then introduced into the cartridge forsample processing. In one embodiment the lysis solution is formulated asshown in Table 13. The proteinase K treated serum/plasma can be mixedwith lysis solution and alcohol in a ratio corresponding to 1.3 mLproteinase K treated serum/plasma, 2.2 mL lysis solution, and 1.5 mlalcohol. In certain embodiments the serum/plasma sample is treated withproteinase K for about 15 minutes. The lysis solution is added cold andheld/mixed for about 10 minutes. Then isopropanol is added to themixture which is then loaded into the cartridge for processing.

As noted above, for serum/plasma the alcohol (e.g., isopropanol)precipitations are typically done at RT, and in particular typically notperformed with “salty” solutions. In certain embodiments longer roomtemperature precipitation times can be used.

TABLE 13 Lysis solution for serum or plasma. Reagent Amount Guanidinethiocyanate (GTC) 4.5M Buffer (e.g., Tris) pH 7.0 45 mM Detergent (e.g.,Tween20)   1% Antifoam SE15 0.01%

In another illustrative, but non-limiting embodiment, a formalin fixedparaffin-embedded (FFPE) sample is prepared by combining the FFPE samplewith proteinase K and a lysis solution comprising a buffer (e.g.,HEPES), a chelator (e.g., EDTA), NaCl, MgCl₂, and optionally sodiumazide and/or an antifoaming agent. The solution is heated (e.g., at 70°C. to 90° C.) for a period of time ranging, for example from about 10minutes up to about 4 hours. An alcohol is added to the solution and thesolution is then introduced into the cartridge for sample processing. Inone embodiment the lysis solution is formulated as shown in Table 14. Inone illustrative, but non-limiting embodiment, 1.2 mL of the lysissolution shown in Table 14 is added to the FFPE section(s). Proteinase Kis added and the mixture is heated, e.g. at 80° C. for about 15 minutes.In certain embodiments heating is performed at 56° C. for 2 hoursfollowed by 90° C. for 30 minutes. Then 1.2 mL of ethanol is added tothe mixture and the mixture is loaded into a sample chamber of thecartridge for processing.

TABLE 14 Lysis buffer for formalin fixed paraffin embedded (FFPE)sample. Tween20   1% NaCl 400 mM  EDTA 25 mM MgCl₂ 10 mM HEPES pH 7.2 50mM Sodium Azide 0.01% SE15 0.01%

Cartridge Operation and Extraction Performance.

When cfDNA is being prepared, in certain embodiments, it is possible toinclude extraction controls to permit monitoring of the quality of theDNA preparation. As illustrated in FIG. 18, there are two different beadsets. One bead set contains an endogenous HMBS primer and probe set fora SAC (sample assay control) and exogenous BG primer and probe set for aSPC (sample prep control). The other contains an endogenous Beta-GlobinPP set for SAC (as well as BG SPC).

It was discovered, inter alia, that the use of GTC in the cartridge maybe less important for serum than plasma samples. Without being bound bya particular theory it is believed that this may be due to the fact thatserum contains less protein. Accordingly, in certain embodiments, thecartridge may contain less GTC or may omit GTC.

FIGS. 19A and 19B show a comparison of the results of cfDNA preparationperformed using a cartridge as described herein compared to the resultsobtained using a conventional “tubefill” procedure. As illustrated inthe qPCR results shown in FIG. 19A, the binding and elution efficienciesobtained using the cartridge are extremely close (within one Ct) tothose obtained using the tubefill protocol. As illustrated in FIG. 19Btitrations of sample concentrations show that the cartridge preparationis conservatively within 1 Ct of the tubefill preparation down to asample concentration as low as about 10 pg. It is believe the cartridgepreparation is even closer to the tubefill protocol at higher sampleconcentrations.

Example 6 Testing a High-Volume Sample Preparation Cartridge

In certain embodiments high volume sample preparation (HSVP) cartridgesare provided for the preparation of large volumes of sample (e.g., up toabout 12 ml to 15 ml). This is particularly useful where the samplecontains DNA at a low concentration (e.g., cfDNA in serum or plasma).One such cartridge is schematically illustrated in FIG. 20A. As showntherein the cartridge provides three chambers (chambers 2, 3, and 5)that can be used to receive a sample. In the illustrated embodiment,each of these chambers can receive about 4 mL of sample and, in certainembodiments, the sample comprises 4 mL of plasma/serum combined with 4mL of GTC and 4 mL of alcohol (e.g., isopropanol).

The sample is introduced into these chambers and the cartridge isoperated as described herein to prepare the sample for PCR and/ormethylation analysis. By way of illustration, in certain embodiments,operation of this cartridge can comprise binding DNA to an affinitycolumn (e.g., for cleanup) and eluting the DNA. In certain embodimentswhere a methylation analysis is to be performed, the operation of thecartridge can further comprise combining the DNA with a conversionreagent (e.g., a bisulfite as described herein) and heating the mixtureto convert the DNA. In certain embodiments, the HSVP cartridge can alsobe configured to desulphonates the converted DNA. In other embodiments,the DNA can be desulphonated in the second (e.g., qPCR) cartridge asschematically illustrated in FIG. 20B. The second cartridge can alsoperform the methylation analysis (e.g. a qPCR analysis).

FIG. 21 shows a comparison of sample preparation results of DNA fromplasma and serum between one cartridge and two cartridge protocols usingthe HMBS or β-globin primer and probe set. As shown therein, there was alinear increase in DNA recovery between 0.5 mL and 4 mL of serum orplasma. Moreover there was little to no loss when using one cartridgefor the preparation and analysis or when using separate cartridges forpreparation and analysis/

Example 7 Optimizing Bisulfite Conversion

In certain embodiments when using a cartridge for a methylation analysisas described herein one potential issue is the optimization of elutionefficiently using the smallest volume possible. Small elution volumesare easier to deal with using spin columns. This problem can beaddressed by using multiple heating steps to process larger samplevolumes.

A second technical concern arises when heating a larger sample (e.g.,minimum 100 μL) when using a smaller (e.g., 50 μL) heating tube orchamber. In certain instances, pressurizations between heating steps canmake it difficult to reproducibly account for volume aspirates anddispenses. Secondly, the absence of pressurization can lead to volumechanges and bubbles especially at higher temperatures. Thirdly, it ispossible to pick up air between heated and unheated samples during portchanges in between heat steps.

To investigate these optimization of bisulfite conversion in a 50 μLtube using single and double heating steps was investigated. Thisexperiment was performed as follows:

Pull 75-80 μL of bisulfite-DNA; heat 95° C.-10s, 65° C.-300s×8;

Pull rest+5-10 μL; pressurize; heat 95° C.-480s, 65° C.-1800s×1.

The results for 0.5 mL of serum are shown in FIG. 22 where the top panelis 1× Heat (converted 33.0, unconverted 34.4) N=4, and the bottom panelis 2× Heat (converted 31.9, unconverted 36.1) N=4.

There is a gain of about 1 Ct in the converted ACTB signal when goingfrom 1× heat to 2× heat. This suggests almost all of the DNA isconverted. This is supported by the fact that there is also a loss ofabout 2 Ct's in the unconverted HMBS signal. A 1 Ct increase is logicalsince we went from heating 50/100 μL to 100/100 μL of DNA-bisulfitesample.

Example 8 Comparison of a DNA Methylation Cartridge with Tube-BasedCommercial Kits

FIG. 23A shows a comparison of the user steps required when performing amethylation analysis using cartridge as described herein (left) ascompared to the steps required when using commercial kits (QIAampMinElute Virus Spin Kit (Qiagen, Inc.), and EZ DNAMethylation-Lightning™ Kit (Zymo Research, Inc.)) to perform the sameanalysis. As can readily be seen the cartridge-based methylationanalysis requires far few user steps with a labor time of about 5minutes as compared to the 2-3 hour labor time required using the kits.

To compare the results produced by the different methods, 200 μL ofserum was purified using the Qiagen kit. The DNA was converted using theZymo kit, purified with a second spin column and eluted with 10 μL. Ranall 10 μL using converted unmethylated ACTB primers and probes (TSR). Incomparison, 200 μL of serum were run in the methylation cartridge asdescribed herein. Results are shown in FIG. 23B. As is readily evident,the cartridge method produced results extremely comparable to thoseobtained using the commercial kits. However, this was accomplished withfar less labor and time.

Example 9 Use of DABSO for DNA Conversion

It was initially attempted to dissolve 5 g DABSO in 5 mL H2O. Ultimatelya few mLs of 10M KOH and a mL of water were added and heated tosolubilize the DABSO and to raise the pH up to between about pH 5 and pH5.5 at an estimated final DABSO concentration of ˜2.5M.

FIG. 24 shows graphs of tubefills of 750 ng of DNA converted using DABSOor the Zymo conversion reagent. The materials were offboard heated (1μg) in a thermocycler and purified with spin columns and run astubefills. The 3 different experiments were:

-   -   1) 120 uL DABSO/30 uL DNA;    -   2) 120 uL Zymo/30 uL DNA; and    -   3) 70 uL Zymo/30 uL DNA (ratio currently in the cartridge).

As shown in FIG. 24, DABSO provided good conversions almost comparableto those obtained using the Zymo reagent.

Example 10 Sensitivity of Detection of Methylated DNA

To evaluate the sensitivity of detection of DNA methylation, convertedACTB gene promoter was detected as a function of copy number using acartridge as described herein. The goal was to detect less than 25copies of converted, unmethylated DNA. As previously shown, falloutswere observed at about 10-50 copies (1 fallout each). Similarsensitivity was observed for methylated DNA targets in a serumbackground.

FIG. 25, panel A, illustrates the detection of methylated DNA in adilution series (MGMT (O-6-Methylguanine-DNA Methyltransferase gene)).As shown therein MGMT was detected down to a level of 78 pg.

The detection of methylated breast cancer markers RASSF1A and AKR1B1 inMBA-453 cells is shown in FIG. 25, panel B. As shown therein, breastcancer markers were detected down to 100 cells.

The detection of methylated pancreatic cancer markers ACTB, BNC1, andADAMTS1 in a dilution series is shown in FIG. 25, panel C. As showntherein, pancreatic markers were detected down to 25 copies.

Table 15 shows the hit rate of pancreatic cancer markers BNC1 andADAMTS1 as a function of concentration. As shown therein these markerscould be detected below 120 pg. Note a positive “hit rate” is anamplification in either gene for a replicate.

Table 15 illustrates the hit rate for pancreatic marker detection as afunction of concentration.

Concentration Hit rate (#/replicates) 0 pg 0/3 16 pg 6/8 30 pg 5/8 60 pg4/8 120 pg 4/4

Example 11 Reverse Complement Multiplex Assay for Both Strands

FIG. 26 illustrates the results for a reverse complement multiplex assayfor both DNA strands. Following bisulfite conversion, both strands losetheir complementarity. Thus, primer and probe sets have to be designedfor one strand or the other, and result in unique amplicons. In additionto providing “more opportunities”, this approach might potentially helpwith sensitivity (at LOD, if only one strand or the other ends up in thetube, this approach would ensure the signal gets picked up).

The multiplex assay allows the multiplex to detect different CpGs at thesame promoter site. The reverse compliment multiplex provides morequeries on target and the possibility to pick up heterogamousmethylation.

Example 12 Detection of DNA Methylation and Mutation in a SingleCartridge

In certain embodiments the multiplex PCR reactions can contain primersand probes that permit the detection of mutations in addition tomethylation in the same cartridge. FIG. 27A illustrates the detection ofmethylated BNC1 and ADAMTS1 along with the KRAS G12D mutation along withcontrol BG (Top Panel) and the detection of methylated BNC1 and ADAMTS1along with the KRAS wildtype along with control BG (Bottom Panel).

FIG. 27B illustrates the simultaneous detection of BNC1 and ADAMTS1methylation in PANC-1 cells (top panel) and MIA-PaCa cells (bottompanel) along with the KRAS G12D mutation.

Example 13 Multiplex Optimization of Pancreatic Cancer

It was determined that methylation analysis of ADAMTS1, BNC1, (andcertain other genes) permits detection and/or staging of pancreaticcancer. Accordingly, the initial multiplex assay for BNC1 and ADAMTS1was optimized to facilitate incorporation of probes for other genes. Tooptimize this assay temperature gradients were run on external andinternal PCRs for forward/reverse bisulfate converted strands.Single-plexes (fwd/rev for each gene) were run at external temperaturesof 56° C., 58° C., and 60° C. and internal temperatures of 64° C., 66°C., and 68° C. (see, e.g., FIG. 28). In certain embodiments the assayswere developed as two 4-plexes for BNC1 and ADAMTS1 and two other genes,one 4-plex for methylation analysis of a forward strand and one 4-plexfor methylation analysis of a reverse strand.

The probes were combined into two sets (see, FIG. 29) based on preferredreaction conditions (salt conditions 40 mM (LS), 60 mM (MS), 80 mM (HS)KCl. 15 mM NH₄SO₄) and optimized for specificity. The final optimizedsalt condition for multiplex 1 was 80 mM KCl, 5 mM MgCl₂, 20 mM Tris pH8.5, and 10 mM NH₄ and for multiplex 2 was 62 mM KCl, 4 mM MgCl₂, 20 mMTris pH 8.5, and 10 mM NH₄.

Example 14 Detection of MGMT Methylation

The O(6)-methylguanine-DNA methyltransferase (MGMT) gene encodes a DNArepair enzyme that can abrogate the effects of alkylating chemotherapysuch as temozolamide. If the MGMT gene is active, the damage is rapidlyrepaired. It is believed that malignant gliomas may have the MGMT geneinactivated due to methylation of its promoter region. Methylated MGMTgene is a predictive initiator for BETTER response to chemotherapy (asthe tumor has no means to repair the DNA damage induced by thealkylating agent).

Primers and probes were developed for the detection of MGMT methylationas illustrated in FIG. 30 and summarized below in Table 16. Inparticular, FIG. 30 illustrates the converted template with CPGs (asdetermined from pyrosequencing) shown in grey. As illustrated afterbisulfate conversion the forward and reverse strand are no longercomplementary permitting separate analysis of each strand.

TABLE 16 Illustrative primer/probe set for detection of MGMT methylation(see, e.g., FIG. 30). Probe SEQ Probe Type Sequence ID NO External 22422MGMT GTTTT(T*)AGAAYG(T*)TTTG 263 Fwd-4 YGTTT 22423 MGMTAAAAAAC(T*)CCRCACTCTTCC 265 Rev-4 Internal 22150 MGMTTTTCGACGTTCGTAGGTTTTCGC 266 Fwd-2 22151 MGMT GCACTCTTCCGAAAACGAAACG 267Rev-2 22419 MGMT Fluor-CCAAACAC(T*)CACCA 268 TaqMan-2 AATC(N*)CAAAC

To evaluate detection sensitivity a MGMT dilution series (5 ng to 78 pgMGMT DNA in a background of 20 ng of HS DNA)) was evaluated using ACTBas a control. In an illustrative experiment, 78 pg of methylated MGMTDNA was only about 10 cycles off the Ct of only unmethylated HS DNA.

As shown in FIG. 31 results produced using the methylation cartridgedescribed herein for the detection of MGMT methylation was compared tothe results produced by pyrosequencing for extracted DNA (FIG. 31, top)and for a FFPE sample (FIG. 31, bottom). Pyrosequencing typically uses acutoff between 10-15% to determine patient stratification. We used anarbitrary cutoff of 12.5 (between ACTB and MGMT) to match pyrosequencingresults as closely as possible. Accordingly, in this example a cutoffwas set at delta Ct=12.5 and calculated concordance with >15%methylation. The cartridge analysis of the extracted DNA show asensitivity of 90% and a specificity at 86% while the cartridge analysisof the FFPE sample showed a sensitivity of 88% and a specificity of 95%.

It is noted that specificity can be improved in two ways: 1) theannealing temperature can be increased as the 62° C. annealingtemperature was rather low. Additionally methylation probes that cover 3(or more) CpGs can be utilized.

Example 15 Detection of BRCA1 Methylation

BRCA1 is a caretaker gene responsible for repairing DNA. It is believethat BRCA1 is involved in homologous, recombination, non-homologous endjoining, and nucleotide excision repair. Women with an abnormal BRCA1gene have an 80% chance of developing breast cancer.

Without being bound to a particular theory, it is believed that BRCA1methylation is a potential predictive marker of response to chemotherapyin triple negative BC patients. Study of NSCLC patient's treated withcisplatin showed those with low BRCA1 expression had improved survivalrates. High levels reduced the effectiveness of chemotherapy byrepairing the damage caused to cancer cells.

In view of these, and other, observations cartridges and methods of usewere developed for detection of BRCA1 methylation. In particular, thePCR condition were optimized as follows: 1) External temperature wasevaluated between 56-62° C. and we settled on a 3 step 56° C. annealingPCR protocol; 2) Internal temperature was evaluated between 64° C.-70°C. and we settled on a two-step 68° C. annealing PCR protocol. Resultsare shown in FIG. 32.

For BRCA1, a one target assay was tested with the ACTB control gene.Eight different cell lines were tested and the effect of adding NH₄ wascompared (see, FIG. 33). BRCA1 methylation was expected to be observedin the 3199 cell line.

Example 16 Detection of Gene Methylation Associated with Lung Cancer

A three target methylation assay for genes whose methylation isassociated with lung cancer (SOX17, CD01, TAC1) was tested along withthe ACTB control gene. The data shown in FIG. 34 indicate that, asexpected, the 3 targets do not come up in a background of normal plasmabut are present to some degree in three different lung cancer celllines.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A set of cartridges for determining the methylation state of anucleic acid, said set of cartridges comprising: a first cartridgecomprising: a sample receiving chamber; a column comprising a firstmatrix material; a temperature controlled channel or chamber; a sampleremoval chamber; and a plurality of chambers containing reagents and/orbuffers, wherein when in use at least one of said chambers contains aconversion reagent; and a second cartridge comprising: a samplereceiving chamber; a column comprising a second matrix material; atemperature controlled channel or chamber; and a plurality of chamberscontaining reagents and/or buffers, wherein when in use at least one ofsaid chambers contains a desulphonation and/or elution reagent.
 2. Theset of cartridges of claim 1, wherein the temperature controlled channelor chamber in said first cartridge is a thermocycling channel orchamber.
 3. The set of cartridges according to claim 1, wherein thetemperature controlled channel or chamber in said second cartridge is athermocycling channel or chamber.
 4. The set of cartridges according toclaim 1, wherein said conversion reagent, when present, comprises acompound selected from the group consisting of ammonium bisulfite,sodium metabisulfite, potassium bisulfite, cesium bisulfite, and DABSO.5. The set of cartridges of claim 4, wherein said conversion reagent,when present, comprises ammonium bisulfite.
 6. The set of cartridgesaccording to claim 1, wherein said conversion reagent, when present, isprovided in a reagent mix comprising scavengers to prevent sulfiteoxidation and/or catalysts.
 7. The set of cartridges of claim 6, whereinsaid conversion reagent, when present, is provided in a reagent mixcomprising scavengers selected from the group consisting of Trolox andhydroquinone.
 8. The set of cartridges according to claim 6, whereinsaid conversion reagent, when present, is provided in a reagent mixcomprising polyamines as catalysts.
 9. The set of cartridges accordingto claim 1, wherein said first cartridge is configured for theconversion reagent to be added to the cartridge by the user.
 10. The setof cartridges according to claim 1, wherein the conversion reagent isprovided as a component in one of said plurality of chambers in saidfirst cartridge.
 11. The set of cartridges according to claim 1, whereinat least one chamber of the plurality of chambers in said secondcartridge contains PCR primers, and/or PCR probes, and/or a PCR mastermix.
 12. The set of cartridges according to claim 1, wherein theplurality of chambers in said second cartridge comprises one or morechambers containing one or more reagents selected from the groupconsisting of methylation specific PCR primers, methylation specific PCRprobes, PCR enzyme(s), and PCR reaction buffer.
 13. The set ofcartridges of claim 12, wherein the plurality of chambers in said secondcartridge comprises at least two chambers containing one or morereagents selected from the group consisting of methylation specific PCRprimers, methylation specific PCR probes, PCR enzyme(s), and PCRreaction buffer.
 14. The set of cartridges according to claim 1, whereinthe plurality of chambers in said second cartridge comprises at leastone chamber containing primers and probes for detection of methylationof a forward strand of a converted DNA.
 15. The set of cartridgesaccording to claim 1, wherein the plurality of chambers in said secondcartridge comprises at least one chamber containing primers and probesfor detection of methylation of a reverse strand of a converted DNA. 16.The set of cartridges according to claim 12, wherein said PCR primers,and/or probes, and/or enzymes are provided as beads.
 17. The set ofcartridges according to claim 1, wherein said second cartridge containsone or more primers shown in Tables 5, 11, or 12, and/or one or moreprobes shown in Tables 5, 11, or
 12. 18. The set of cartridges of claim17, wherein said second cartridge contains the following probes andprimers for determining methylation of MGMT using a nested PCR reaction:an external forward primer (248b) comprising the nucleotide sequenceGTTTT(T*)AGAAYG(T*)TTTGYGTTT (SEQ ID NO:263); an external reverse primer(249b) comprising the nucleotide sequence: AAAAAAC(T*)CCRCACTCTTCC (SEQID NO:265); an internal forward primer (250) comprising the nucleotidesequence TTTCGACGTTCGTAGGTTTTCGC (SEQ ID NO:266); an internal reverseprimer (251) comprising the nucleotide sequence GCACTCTTCCGAAAACGAAACG(SEQ ID NO:267); and a probe (252a) comprising the nucleotide sequencefluor-CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker (SEQ ID NO:268).
 19. Theset of cartridges according to claim 17, wherein said second cartridgecontains the following probes and primers for determining methylation ofACTB using a nested PCR reaction: an external forward primer (102)comprising the nucleotide sequence GTGATGGAGGAGGTTTAGTAAGTT (SEQ IDNO:103); an external reverse primer (103) comprising the nucleotidesequence CCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO:104); an internal forwardprimer (148) comprising the nucleotide sequenceGGTTTAGTAAGTTTTTTGGATTGTG (SEQ ID NO:149); an internal reverse primer(149) comprising the nucleotide sequence CCTTAAAAATTACAAAAACCACAAC (SEQID NO:150); and a probe (178) comprising the nucleotide sequencefluor-CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker (SEQ ID NO:179).
 20. Asystem for determining the methylation of a nucleic acid in a biologicalsample, said system comprising: an enclosure configured to contain oneor more sample processing modules, each sample processing moduleconfigured to hold a removable cartridge first cartridge and/or secondcartridge of said set of cartridges according to claim 1; where saidsystem is configured to: operate the sample processing modules toperform sample processing; operate the first cartridge of said set ofcartridges to perform a bisulfite conversion of a nucleic acid in asample introduced into said first cartridge; and/or perform adesulphonation and to determine methylation of one or more targetnucleic acids within a corresponding removable sample cartridge.
 21. Amethod of determining the methylation state of a nucleic acid, saidmethod comprising: providing a biological sample in a sample chamber ofa first cartridge in a set of cartridges according to claim 1; andoperating said first cartridge to: bind DNA in said sample to said firstmatrix material; wash the bound DNA; elute the bound DNA off of thematrix material; combine the eluted DNA with said conversion reagent;heat the mixture of DNA and conversion reagent in said temperaturecontrolled channel or chamber perform a bisulfite conversion of saidDNA; and deliver the converted DNA into the sample removal chamber ofsaid first cartridge.
 22. The method of claim 21, wherein said methodfurther comprises: providing converted DNA in a sample chamber of asecond cartridge in a set of cartridges, said set of cartridgescomprising: a first cartridge comprising: a sample receiving chamber; acolumn comprising a first matrix material; a temperature controlledchannel or chamber; a sample removal chamber; and a plurality ofchambers containing reagents and/or buffers, wherein when in use atleast one of said chambers contains a conversion reagent; and the secondcartridge comprising: a sample receiving chamber; a column comprising asecond matrix material; a temperature controlled channel or chamber; anda plurality of chambers containing reagents and/or buffers, wherein whenin use at least one of said chambers contains a desulphonation and/orelution reagent; and operating said second cartridge to: bind saidconverted DNA to said second matrix material; wash the bound convertedDNA; elute the washed converted DNA from said second matrix material;and desulphonate the converted DNA.
 23. The method of claim 22, whereinsaid second cartridge is operated to elute the converted DNA from saidsecond matrix material before desulphonation.
 24. The method of claim22, wherein said second cartridge is operated to elute the converted DNAfrom said second matrix material after or during desulphonation.
 25. Themethod according to claim 22, wherein said method comprises operatingsaid second cartridge to perform methylation specific PCR, and/ornucleic acid sequencing, and/or high resolution melting analysis (HRM)on said converted DNA to determine the methylation of said DNA.
 26. Amethod of detecting a cancer or the predisposition to a cancer in asubject, said method comprising: providing a biological sample from saidsubject, wherein said biological sample comprises a DNA; utilizing a setof cartridges according to claim 1, wherein said first cartridge of saidset of cartridges is used to perform a bisulfite conversion of said DNA;and said second cartridge of said set of cartridges is used todesulphonate the converted DNA and to detect methylation of one or moregene promoters in said DNA whose methylation state is a marker for acancer, where an increase in methylation of said one or more genepromoters is indicative of the presence of a cancer or a predispositionto a cancer or a stage of a cancer or precancer.
 27. A kit for thedetermination of DNA methylation, said kit comprising: a containercontaining a first cartridge and/or a second cartridge of a set ofcartridges according to claim
 1. 28. The kit according to claim 27,wherein said kit comprises a conversion reagent in said cartridge or ina container separate from the cartridge.
 29. The kit of claim 28,wherein said kit comprises said conversion reagent in a containerseparate from the cartridge.
 30. The kit of claim 28, wherein said kitcomprises said conversion reagent in a chamber of the cartridge.
 31. Thekit according to claim 28, wherein said conversion reagent comprises acompound selected from the group consisting of sodium metabisulfite,potassium bisulfite, cesium bisulfite, ammonium bisulfite, and DABSO.